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Section 5.

2Purification of Cells and Their Parts

Most animal and plant tissues contain a mixture of cell types. However, an investigator often
wishes to study a pure population of one type of cell. In some cases, cells differ in some physical
property that allows different cell types to be separated. White blood cells (leukocytes) and red
blood cells (erythrocytes), for instance, have very different densities because erythrocytes have
no nucleus; thus these cells can be separated on the basis of density. Since most cell types cannot
be differentiated so easily, other cell-separation techniques have had to be developed. Similarly,
it is essential to isolate quantities of each of the major subcellular organelles to study their
structures and metabolic functions in detail.
Go to:
Flow Cytometry Separates Different Cell Types
A flow cytometer can identify different cells by measuring the light they scatter, or the
fluorescence they emit, as they flow through a laser beam; thus it can sort out cells of a particular
type from a mixture. Indeed, a fluorescence-activated cell sorter (FACS), an instrument based on
flow cytometry, can select one cell from thousands of other cells (Figure 5-21). For example, if
an antibody specific to a certain cell-surface molecule is linked to a fluorescent dye, any cell
bearing this molecule will bind the antibody and will then be separated from other cells when it
fluoresces in the FACS. Once sorted from the other cells, the selected cell can be grown in

Figure 5-21
Fluorescence-activated cell sorter (FACS). A concentrated suspension of cells is allowed to react
with a fluorescent antibody or a dye that binds to a particle or molecule such as DNA.
The (more...)
This procedure is commonly used to purify the different types of white blood cells, each

of which bears on its surface one or more distinctive proteins and thus will bind monoclonal
antibodies specific for that protein. Such FACS separations are more difficult to conduct on
cultured cells or cells from animal tissues, which interact with adjacent cells and are surrounded
by an extracellular matrix. Samples must be treated with proteases to degrade the extracellularmatrix proteins and cell-surface proteins that attach cells in tissues to one another; these
proteases usually also degrade the distinctive cell-surface marker proteins that distinguish one
cell type from another.

Other uses of flow cytometry include the measurement of a cells DNA and RNA content and
the determination of its general shape and size. The FACS can make simultaneous measurements
of the size of a cell (from the amount of scattered light) and the amount of DNA it contains (from
the amount of fluorescence from a DNA-binding dye).
Go to:
Disruption of Cells Releases Their Organelles and Other Contents
The initial step in purifying subcellular structures is to rupture the plasma membrane and the cell
wall, if present. First, the cells are suspended in a solution of appropriate pH and salt content,
usually isotonic sucrose (0.25 M) or a combination of salts similar in composition to those in the
cells interior. Many cells can then be broken by stirring the cell suspension in a high-speed
blender or by exposing it to highfrequency sound (sonication). Plasma membranes can also be
sheared by special pressurized tissue homogenizers in which the cells are forced through a very
narrow space between the plunger and the vessel wall. Generally, the cell solution is kept at 0 C
to best preserve enzymes and other constituents after their release from the stabilizing forces of
the cell.
Because the plasma membrane is highly permeable to water but poorly permeable to the salts
and other small molecules (solutes) within cells, osmotic flow can be enlisted to help rupture
cells. Recall that water flows across a semipermeable membrane, such as the plasma membrane,
from a solution of high water (low solute) concentration to one of low water (high solute)
concentration until the water concentration on both sides is equal. Consequently, when cells are
placed in ahypotonic solution (i.e., one with a lower salt concentration than that of the cell
interior), water flows into the cells (Figure 5-22). This osmotic flow causes the cells to swell and
then more easily rupture. Conversely, in a hypertonicsolution (i.e., one with a higher salt
concentration than that of the cell interior), water flows out of cells, causing them to shrink.
When cells are placed in an isotonic solution (i.e., one with a salt concentration equal to that of
the cell interior), there is no net movement of water in or out of cells. For this reason,
an isotonic solution is best for preserving normal cell structure.

Figure 5-22

Response of animal cells to the osmotic strength of the surrounding medium. Sodium, potassium,
and chloride ions do not move freely across the cell membrane, but water does. (a) When
the (more...)
Disrupting the cell produces a mix of suspended cellular components, the homogenate, from
which the desired organelles can be retrieved. Because rat liver contains an abundance of a single
cell type, this tissue has been used in many classic studies of cell organelles. However, the same
isolation principles apply to virtually all cells and tissues, and modifications of these cellfractionation techniques can be used to separate and purify any desired components.
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Different Organelles Can Be Separated by Centrifugation
In Chapter 3 we discussed the principles of centrifugation and the uses of centrifugation
techniques for separating proteins and nucleic acids. Similar approaches are used for separating
and purifying the various organelles, which differ in both size and density.
Most fractionation procedures begin with differential centrifugation at increasingly higher speeds
(Figure 5-23), also called differential-velocity centrifugation. The different sedimentation rates of
various cellular components make it possible to separate them partially by centrifugation. Nuclei
and viral particles can sometimes be purified completely by such a procedure. After
centrifugation at each speed for an appropriate time, the supernatant is poured off and
centrifuged at higher speed. Each pelleted fraction can be resuspended and further separated by
equilibrium densitygradient centrifugation (discussed next).

Figure 5-23
Cell fractionation by differential centrifugation. Generally, the cellular homogenate is first
filtered or centrifuged at relatively low speeds to remove unbroken cells. Then
centrifugation (more...)
Differential centrifugation does not yield totally pure organelle fractions. One method for further
purifying fractions isequilibrium density-gradient centrifugation, which separates cellular
components according to their density. The impure organelle fraction is layered on top of a
solution that contains a gradient of a dense nonionic substance, such as sucrose or glycerol. The
tube is centrifuged at a high speed (about 40,000 rpm) for several hours, allowing each particle to
migrate to an equilibrium position where the density of the surrounding liquid is equal to the
density of the particle. In typical preparations from animal cells, the rough endoplasmic

reticulum (density=1.20 g/cm3) separates well from the Golgi vesicles (density=1.14 g/cm3)
and from the plasma membrane (density=1.12 g/cm3). (The higher density of the rough
endoplasmic reticulum is due largely to the ribosomes bound to it.) This method also works well
for resolving lysosomes, mitochondria, and peroxisomes in the initial mixed fraction obtained by
differential centrifugation (Figure 5-24).

Figure 5-24
Separation of organelles from rat liver by equilibrium density-gradient centrifugation. For
example, the material deposited as a pellet by centrifugation at 15,000 g (see Figure 5-23)
can (more...)
Since each organelle has unique morphological features, the purity of organelle preparations can
be assessed by examination in an electron microscope (Figure 5-25). Alternatively, organellespecific marker molecules can be quantified. For example, the protein cytochrome c is present
only in mitochondria, so the presence of this protein in a fraction of lysosomes would indicate its
contamination by mitochondria. Similarly, catalase is present only in
peroxisomes; acid phosphatase, only in lysosomes; and ribosomes, only in the
rough endoplasmic reticulum or thecytosol.

Figure 5-25
Electron micrographs of purified rat liver organelles. Seen here are (a) nuclei, (b) rough
endoplasmic reticulum, sheared into smaller vesicles termedmicrosomes, and (c) peroxisomes.
Note (more...)
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Organelle-Specific Antibodies Are Useful in Preparing Highly Purified Organelles
Cell fractions often contain more than one type of organelle even after differential and
equilibrium density-gradient centrifugation. Such fractions can be further purified by
immunological techniques, using monoclonal antibodies for various organellespecific membrane proteins. One example is the purification of a particular class of cellular
vesicles whose outer surface is coated with the protein clathrin (Figure 5-26). An antibody to
clathrin, bound to a bacterial carrier, can selectively bind these vesicles in a crude preparation of
membranes, and the whole antibody complex can then be isolated by low-speed centrifugation. A

recently developed technique uses tiny metallic beads coated with specific antibodies. Organelles
that bind to the antibodies, and thus are linked to the metallic beads, are recovered from the
preparation by adhesion to a small magnet on the side of the test tube.

Figure 5-26
Immunological purification of clathrin-coated vesicles. (a) A suspension of membranes from rat
liver is incubated with an antibody specific for clathrin, a protein that coats the outer
surface (more...)
All cells contain a dozen or more different types of small membrane-limited vesicles of about the
same size (50100 nm in diameter) and density. Because of their similar size and density, these
vesicles are difficult to separate from one another by centrifugation techniques. Immunological
techniques are particularly useful for purifying specific classes of such vesicles. Fat and muscle
cells, for instance, contain a particular glucose transporter (GLUT4) that is localized to the
membrane of a specific kind of vesicle. When insulin is added to the cells, these vesicles fuse
with the cell-surface membrane, a process critical to maintaining the appropriate concentration of
sugar in the blood (see Figure 5-7). These vesicles can be purified using an antibody that binds to
a segment of the GLUT4 protein that faces the cytosol.
Go to:

Flow cytometry can identify different cells based on the light they scatter or the
fluorescence they emit. The fluorescence-activated cell sorter (FACS) is particularly
useful in separating different types of white blood cells (see Figure 5-21). A
cells DNA and RNA content also can be measured with a FACS.

Disruption of cells by vigorous homogenization, sonication, or other techniques releases

their organelles. When placed in a hypotonic solution, cells swell, thereby weakening
the plasma membrane and making it easier to rupture.

Sequential differential-velocity centrifugation of a cell homogenate yields partially

purified organelles that differ in mass (see Figure 5-23).

Equilibrium density-gradient centrifugation, which separates cellular components

according to their density, can further purify cell fractions obtained by differential

Because the membrane surrounding each type of organelle contains organelle-specific

proteins, immunological techniques are very useful in purifying organelles and vesicles,
particularly those that have a similar size and density.

Figure 5-22Response of animal cells to the osmotic strength of the surrounding


Sodium, potassium, and chloride ions do not move freely across the cell membrane, but
water does. (a) When the medium is isotonic, there is no net flux of water into or out of
the cell. (b) When the medium is hypotonic, water flows into the cell (red arrow) until the
ion concentration inside and outside the cell is the same. Here, the initial cytosolic ion
concentration is twice the extracellular ion concentration, so the cell tends to swell to
twice its original volume, at which point the internal and external ion concentrations are
the same. (c) When the medium is hypertonic, water flows out of the cell until the ion
concentration inside and outside the cell is the same. Here, the initial cytosolic ion

concentration is half the extracellular ion concentration, so the cell is reduced to about
half its original volume.

Figure 5-23Cell fractionation by differential centrifugation

Generally, the cellular homogenate is first filtered or centrifuged at relatively low speeds
to remove unbroken cells. Then centrifugation of the homogenate at a slightly faster
speed or for a longer duration will selectively pellet the nucleusthe
largest organelle (usually 510 m in diameter). A centrifugal force of 600 g (600 times
the force of gravity) is necessary to sediment nuclei; this is generated by a typical
centrifuge rotor operating at 500 revolutions per minute (rpm). The undeposited material
(the supernatant) is next centrifuged at a higher speed (15,000 g5 min), which deposits
themitochondria, chloroplasts, lysosomes,and peroxisomes. A subsequent centrifugation
in the ultracentrifuge (100,000 g60 min) results in deposition of the plasma membrane,
fragments of the endoplasmic reticulum, and large polyribosomes. A force of
100,000 g requires about 50,000 rpm in an ultracentrifuge; at this speed, the rotor
chamber is kept in a high vacuum to reduce heating due to friction between air and the
spinning rotor. The recovery of ribosomal subunits, small polyribosomes, and particles
such as complexes of enzymes requires additional centrifugation at still higher speeds.

Only the cytosolthe soluble aqueous portion of the cytoplasmremains undeposited

after centrifugation at 300,000 g for 2 hours.

Figure 5-24Separation of organelles from rat liver by equilibrium density-gradient


For example, the material deposited as a pellet by centrifugation at 15,000 g (see Figure
5-23) can be resuspended and layered on a density gradient composed of layers of
increasingly more dense sucrose solutions in a centrifuge tube. Under centrifugation,
each organelle migrates to its appropriate equilibrium density and remains there. To
obtain a good separation of lysosomes from mitochondria, the liver is perfused with a
solution containing a small amount of detergent before the tissue is disrupted. The
detergent is taken into the cells by endocytosis and transferred to the lysosomes, making
them less dense than they would normally be, thereby affording a clean separation from
the mitochondria.

Molisch's Test - Qualitative Test in

Molisch's test is a test for carbohydrates or compounds which can
be dehydrated to furfural or furfural derivatives in the presence of
the concentrated sulphuric acid (H2SO4). Furfural is derived from

the dehydration and rearrangement of pentoses and pentosans

while hydroxymethyl is produced from hexoses and hexosans.
The -naphthol reacts with the cyclic aldehydes to form purple
coloured condensation products.
Although this test will detect compounds other than carbohydrates
(i.e.) glycoproteins, a negative result indicates the absence of

Biuret's Test - Qualitative Test in Proteins

Biuret's test for peptide bonds is a reaction characterised by a

violet colour upon the addition of copper sulphate to all
compounds with two amide or peptide bonds linked directly or
through an intermediate carbon atom. It is used in the detection
and estimation of proteins and peptides having more than two
A pink to purple/violet colour indicates the presence of proteins. A
more intense coloration indicates the presence of more complex

The Molisch Test

Shows positive test for:

All carbohydrates. Monosaccharides give a rapid positive test.

Disaccharides and polysaccharides react slower.
The test reagent dehydrates pentoses to form furfural (top
reaction) and dehydrates hexoses to form 5-hydroxymethyl
furfural (bottom reaction). The furfurals further react with naphthol present in the test reagent to produce a purple
product (reaction not shown).

How to perform the test:

Two ml of a sample solution is placed in a test tube. Two drops
of the Molisch reagent (a solution of -napthol in 95% ethanol)
is added. The solution is then poured slowly into a tube
containing two ml of concentrated sulfuric acid so that two
layers form.
A positive test is indicated by:

The formation of a purple product at the interface of the two


a negative test (left) and a positive test (right)
The Biuret Test

Shows positive test for:

Biuret and proteins.
How to perform the test:
Prepare a solution or suspension of the sample by placing ~0.2
g in 10 ml of water. Ten drops of 1.5 M NaOH (a colorless
solution) and 2 drops of 0.1 M CuSO4 (a light blue solution) are
A positive test is indicated by:
a deep blue/purple color due to the copper ion complex with
the amide group of the protein.

a negative test (left) and a positive test (right)
1.What is diphenylamine ?
Diphenylamine is also known as Diphenylamine redox, with its CAS No. 122-39-4, and it has the
molecular formula of C12H11N and a molecular weight of 169.22. The other properties of diphenylamine
include: Density: 1.16; Melting point: 52-54 C; Boiling point: 302 C; Flash point : 152 C; Water
solubility: Slightly soluble. 0.03 g/100 mL. Diphenylamine is often used for producing stabilizer of
explosive and fuel.
2. Diphenylamine test
One of the suppliers of diphenylamine, Hangzhou Chemfar Ltd., which has more than 20 years'
experience focused on providing integrated development and manufacturing service to the
pharmaceutical and fine chemical industry, showed that diphenylamine was of great importance in
many experiments.
For example, in the identification of DNA, DNA can be identified chemically with the dische
diphenylamine test. One major difference between DNA and RNA is their sugar: DNA contains
deoxyribose, whereas RNA contains ribose. This is what is diphenylamine test for deoxyribose for. In
this process, the reaction between the Dische reagent and 2-deoxypentose results in the development
of a blue color. The reaction depends on the conversion of the pentose to w-hydroxylaevulinic aldehyde
which then reacts with diphenylamine to give a a blue colored complex. The intensity of the blue color
is proportional to the concentration of DNA. Dische reagent does not react with the ribose sugar in RNA
and does not form a blue-colored complex. Dische diphenylamine test -- A chemical test utilized to
detect the presence of DNA in a substance.

Five questions about Diphenylamine

Posted on September 27, 2013by bbz2013

1.Diphenylamine test for deoxyribose?

When adding diphenylamine to deoxyribose you will get a dark deep purple color. If you
react diphenylamine with crude DNA you will get a pink-violet color. DIphenylamine
test is quantitative and the darker the color the greater the concentration of DNA in the
2.What is diphenylamine test for deoxyribose for?
It can be used to test for the presence of DNA in a substance. The test consists of
diphenylamine(CAS NO.: 122-39-4) in acid, which reacts with the deoxyribose in the
DNA to produce a blue color. The intensity of the blue color is in direct proportion to the
concentration of DNA.
One major difference between DNA and RNA is their sugar: DNA contains deoxyribose,
whereas RNA contains ribose. DNA can be identified chemically with the Dische
diphenylamine test. The reaction between the Dische reagent and 2-deoxypentose
results in the development of a blue color. The reaction depends on the conversion of
the pentose to w-hydroxylaevulinic aldehyde which then reacts with diphenylamine to
give a a blue colored complex. The intensity of the blue color is proportional to the
concentration of DNA. Dische reagent does not react with the ribose sugar in RNA and
does not form a blue-colored complex
3.What is responsible for the formation of the absorbed color of DNA with
Answer 3:
When you heat DNA in acid the 2-deoxyribose is converted to w-hydroxylevulinyl

aldehyde, which reacts with the compound, diphenylamine, to produce a blue-colored

4.Why does blue colour appears in diphenylamine test?
Answer 4:
Blue color appears in diphenylamine test because of the reaction of nitrates.
5.Detection of DNA with Diphenylamine?
Answer 5:
A diphenylamine indicator is often used for the detection of DNA. Diphenylamine is
an organic compound and classified as an aromatic amine due to the presence of a
benzene ring in its chemical makeup. Chemical hydrolysis of DNA must take place when
looking for it using diphenylamine. The DNA concentration in an organism is measured
by looking for the absorbency rate of the diphenylamine. DNA molecules are the
building blocks of life and contain specific genetic coding for the organism it is
contained in.
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Feulgen's test
A Dictionary of Biology | 2004 | 486 words | Copyright

Feulgen's test A histochemical test in which the distribution of DNA in the chromosomes of dividing cell
nuclei can be observed. It was devised by the German chemist R. Feulgen (18841955). A tissue section
is first treated with dilute hydrochloric acid to remove the purine bases of the DNA, thus exposing the
aldehyde groups of the sugar deoxyribose. The section is then immersed inSchiff's reagent, which
combines with the aldehyde groups to form a magenta-coloured compound.

Feulgen stain
From Wikipedia, the free encyclopedia

Black and white microphotograph ofFeulgen stained, intact tick salivary glands infected by deer tick
virus.Hypotrophied salivary acinus filled with amorphous masses of pinkstaining (=Feulgen positive) material
(arrows). Scale bar = 10 m.

Feulgen stain is a staining technique discovered by Robert Feulgen and used in histology to
identify chromosomal material or DNA in cell specimens. It depends on acid hydrolysis of DNA,
therefore fixating agents using strong acids should be avoided.
The specimen is subjected to warm (60 C) hydrochloric acid, then to Schiff reagent. In the past,
a sulfite rinse followed, but this is now considered unnecessary. Optionally, the sample can
be counterstained with Light Green SF yellowish. Finally, it is dehydrated withethanol, cleared
with xylene, and mounted in a resinous medium.
DNA should be stained red. The background, if counterstained, is green.
The Feulgen reaction is a semi-quantitative technique. If the only aldehydes remaining in the cell are
those produced from the hydrolysisof DNA, then the technique is quantitative for DNA. It is possible
to use an instrument known as a microdensitometer or microspectrophotometer to actually measure
the intensity of the pink Feulgen reaction for a given organelle. Using this procedure, it was early
determined that interphase cells were composed of two populations, those with diploid DNA and
those with tetraploid DNA (two complete genomes). The nuclei looked identical, but one contained
twice as much DNA. This gave rise to the division of the interphase period of the cell cycle to G1, S,
and G2 phases based on the synthesis of that extra DNA.[1]

Bial's test
From Wikipedia, the free encyclopedia

Bial's test


Colorimetric method



Bial's test is a chemical test for the presence of pentoses. It is named after Manfred Bial, German
Physician. The components include orcinol, hydrochloric acid, and ferric chloride. A pentose, if
present, will be dehydrated to form furfural which then reacts with the orcinol to generate a colored
substance. The solution will turn bluish and a precipitate may form. The solution shows
twoabsorption bands, one in the red between Fraunhofer lines B and C and the other near the D
line.[1] An estimate of the relevantwavelengths can be made by referring to the Fraunhofer
lines article.


Bial's reagent consists of 0.4g orcinol, 200 ml of concentrated hydrochloric acid and 0.5 ml of a 10%
solution of ferric chloride.[2] Bial's test is used to distinguish pentoses from hexoses; this distinction is
based on the color that develops in the presence of orcinol and iron (III) chloride. Furfural from
pentoses gives a blue or green color. The relatedhydroxymethylfurfural from hexoses may give a
muddy-brown or gray solution, but this is easily distinguishable from the green color of pentoses.

Quantitative version[edit]
The test may be performed as a quantitative colorimetric test using a spectrophotometer. Fernell and
King have published a procedure for simultaneous determination of pentoses and hexoses from
measurements at two wavelengths [3] Various versions of this test are widely used for a quick
chemical determination of RNA; in this context it is usually called the orcinol test.

Molisch's test

From Wikipedia, the free encyclopedia

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and removed. (December 2014)

Molisch's test

Colorimetric method



Molisch's test (named after Austrian botanist Hans Molisch) is a sensitive chemical test for the
presence of carbohydrates, based on the dehydration of the carbohydrate by sulfuric acid or
hydrochloric acid to produce an aldehyde, which condenses with two molecules of phenol (usually naphthol, though other phenols (e.g. resorcinol, thymol) also give colored products), resulting in a
red- or purple-colored compound.

1 Procedure

2 Reaction

3 References

4 See also

The test solution is combined with a small amount of Molisch's reagent (-naphthol dissolved in
ethanol) in a test tube. After mixing, a small amount of concentrated sulfuric acidis slowly added
down the sides of the sloping test-tube, without mixing, to form a layer. A positive reaction is
indicated by appearance of a purple ring at the interface between the acid and test layers.

All carbohydrates monosaccharides, disaccharides, and polysaccharides should give a positive
reaction, and nucleic acids and glycoproteins also give a positive reaction, as all these compounds
are eventually hydrolyzed to monosaccharides by strong mineral acids. Pentoses are then
dehydrated to furfural, while hexoses are dehydrated to 5-hydroxymethylfurfural. Either of these
aldehydes, if present, will condense with two molecules of naphthol to form a purple-colored product,
as illustrated below by the example ofglucose:

Biuret test
From Wikipedia, the free encyclopedia

The characteristic color of a positive biuret test

The biuret test is a chemical test used for detecting the presence of peptide bonds. In the presence
of peptides, a copper(II) ion formsviolet-colored coordination complexes in an alkaline solution.
Several variants on the test have been developed, such as the BCA test and the Modified Lowry

The biuret reaction can be used to assess the concentration of proteins because peptide bonds
occur with the same frequency per amino acid in the peptide. The intensity of the color, and hence
the absorption at 540 nm, is directly proportional to the protein concentration, according to the BeerLambert law.
Despite its name, the reagent does not in fact contain biuret ((H2N-CO-)2NH). The test is so named
because it also gives a positive reaction to the peptide-like bonds in the biuret molecule.

1 Procedure

2 Biuret reagent

3 High sensitivity variants of the biuret test

4 References

5 External links and notes

An aqueous sample is treated with an equal volume of 1% strong base (sodium or potassium
hydroxide most often) followed by a few drops of aqueous copper(II) sulfate. If the solution turns
purple, protein is present. 5160 mg/mL can be determined. A peptide of a chain length of at least 3
amino acids is necessary for a significant, measurable color shift with these reagents. [3]

Biuret reagent[edit]
The Biuret reagent is made of sodium hydroxide (NaOH) and hydrated copper(II) sulfate, together
with potassium sodium tartrate.[4] Potassium sodium tartrate[5] is added to complex to stabilize the
cupric ions. The reaction of the cupric ions with the nitrogen atoms involved in peptide bonds leads
to the displacement of the peptide hydrogen atoms under the alkaline conditions. A tri or tetra
dentate chelate of with the peptide nitrogen produces the "buret" color. This is found with dipeptides
(Datta,S.P., Leberman,R., and Rabin,B.R., Trans.Farad.Soc. (1959), 55, 2141.)

Reaction of proteins with Cu2+, forming a complex containing Cu+

The reagent is commonly used in the biuret protein assay, a colorimetric test used to determine
protein concentration by UV/VIS spectroscopy at wavelength 565 nm.

High sensitivity variants of the biuret test[edit]

Two major modifications of the biuret test are commonly applied in modern colorimetric analysis of
peptides: the bicinchoninic acid (BCA) assay and the Lowry assay. In these tests, the Cu + formed
during the biuret reaction reacts further with other reagents, leading to a deeper color.
In the BCA test, Cu+ forms a deep purple complex with bicinchoninic acid (BCA),[6] which absorbs
around 562 nm, producing the signature violet color. The water-soluble BCA/copper complex
absorbs much more strongly than the peptide/copper complex, increasing the sensitivity of the biuret
test by a factor of around 100: the BCA assay allows to detect proteins in the range of 0.0005 to
2 mg/mL). Additionally, the BCA protein assay gives the important benefit of compatibility with
substances like up to 5% surfactants in protein samples.
In the Lowry protein assay Cu+ is oxidized back to Cu2+ by MoVI in Folin-Ciocalteu's reagent, which
forms molybdenum blue (MoIV). Tyrosine residues in the protein also form molybdenum blue under
these circumstances. In this way, proteins can be detected in concentrations between 0.005 and
2 mg/mL.[7] Molybdenum blue in turn can bind certain organic dyes such as malachite
green andAuramin O, resulting in further amplification of the signal.[8]

Sudan IV
From Wikipedia, the free encyclopedia

Sudan IV

Systematic IUPAC name
1-(2-methyl-4-(2-methylphenyldiazenyl) phenyl)
Other names
Sudan R, C.I. Solvent Red 24, C.I. 26105, Lipid Crimson,
Oil Red, Oil Red BB, Fat Red B, Oil Red IV, Scarlet Red,
Scarlet Red N.F, Scarlet Red Scharlach, Scarlet R
CAS Registry Number




Jmol-3D images




Chemical formula


Except where otherwise noted, data are given for

materials in their standard state (at 25 C [77 F],
100 kPa).

verify (what is:

/ ?)

Infobox references

Sudan IV (C24H20N4O) is a lysochrome (fat-soluble dye) diazo dye used for

the staining of lipids, triglycerides and lipoproteins on frozen paraffin sections. It has the appearance
of reddish brown crystals with melting point 199 C and maximum absorption at 520(357) nm.
Sudan IV is one of the dyes used for Sudan staining. Similar dyes include Oil Red O, Sudan III,
and Sudan Black B. Staining is an important biochemical technique, offering the ability to visually
qualify the presence of the fatty compound of interest without isolating it. For staining purposes
Sudan IV can be made up in propylene glycol[1]. Alternatively, authors have reported using the dye
saturated in isopropyl alcohol, 95% ethanol, or 0.05% by weight in acetone:ethanol:water (50:35:15)
[citation needed]
. The idea is to use a moderately apolar solvent to solubilize the dye allowing it to partition
into the highly apolar fat without the solvent solubilizing the fat to be stained.
Sudan I, Sudan III, and Sudan IV have been classified as category 3 carcinogens by
the International Agency for Research on Cancer.[1]
In its purified form it is called Biebrich scarlet R, which should not be confused with the watersoluble Biebrich scarlet.
In industry, it is used to color nonpolar substances like oils, fats, waxes, greases,
various hydrocarbon products, and acrylicemulsions. Sudan IV is also used in United Kingdom as
a fuel dye to dye lower-taxed heating oil; because of that it is also known asOil Tax Red. As a food
dye, Sudan IV is considered an illegal dye, mainly because of its harmful effect over a long period of
time, as it is a carcinogen. It was ruled unsafe in the 1995 food safety regulations report.

From Wikipedia, the free encyclopedia

For specific types of electrophoresis (for example, the process of administering

medicine, iontophoresis), see Electrophoresis (disambiguation).

Illustration of electrophoresis

Illustration of electrophoresis retardation

Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a
spatially uniform electric field.[1][2][3][4][5][6] This electrokinetic phenomenon was observed for the first time
in 1807 by Ferdinand Frederic Reuss (Moscow State University),[7] who noticed that the application of
a constant electric field caused clay particles dispersed in water to migrate. It is ultimately caused by
the presence of a charged interface between the particle surface and the surrounding fluid. It is the
basis for a number of analytical techniques used in biochemistry for separating molecules by size,
charge, or binding affinity.
Electrophoresis of positively charged particles (cations) is called cataphoresis, while
electrophoresis of negatively charged particles (anions) is called anaphoresis. Electrophoresis is a
technique used in laboratories in order to separate macromolecules based on size. The technique
applies a negative charge so proteins move towards a positive charge. This is used for both DNA
and RNA analysis. Polyacrylamide gel electrophoresis (PAGE) has a clearer resolution than agarose
and is more suitable for quantitative analysis. In this technique DNA foot-printing can identify how
proteins bind to DNA. It can be used to separate proteins by size, density and purity. It can also be

used for plasmid analysis, which develops our understanding of bacteria becoming resistant to

Gel electrophoresis
From Wikipedia, the free encyclopedia

Gel electrophoresis

Gel electrophoresis apparatus An agarose gel is placed

in this buffer-filled box and an electrical field is applied
via the power supply to the rear. The negative terminal
is at the far end (black wire), so DNA migrates toward
the anode (red wire).



Other techniques


Capillary electrophoresis
Two-dimensional gel electrophoresis
Temperature gradient gel


Digital image of 3 plasmid restriction digests run on a 1% w/v agarose gel, 3 volt/cm, stained with ethidium
bromide. The DNA size marker is a commercial 1 kbp ladder. The position of the wells and direction of DNA
migration is noted.

Gel electrophoresis is a method for separation and analysis of macromolecules

(DNA, RNA and proteins) and their fragments, based on their size and charge. It is used in clinical
chemistry to separate proteins by charge and/or size (IEF agarose, essentially size independent)
and in biochemistry and molecular biology to separate a mixed population
of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to
separate proteins by charge.[1]
Nucleic acid molecules are separated by applying an electric field to move the negatively charged
molecules through a matrix ofagarose or other substances. Shorter molecules move faster and
migrate farther than longer ones because shorter molecules migrate more easily through the pores
of the gel. This phenomenon is called sieving.[2]
Proteins are separated by charge in agarose because the pores of the gel are too large to sieve
Gel electrophoresis can also be used for separation of nanoparticles.
Gel electrophoresis uses a gel as an anticonvective medium and/or sieving medium during
electrophoresis, the movement of a charged particle in an electrical field. Gels suppress the thermal
convection caused by application of the electric field, and can also act as a sieving medium,

retarding the passage of molecules; gels can also simply serve to maintain the finished separation,
so that a post electrophoresis stain can be applied. [3] DNA Gel electrophoresis is usually performed
for analytical purposes, often after amplification of DNA via PCR, but may be used as a preparative
technique prior to use of other methods such as mass spectrometry,RFLP, PCR, cloning, DNA
sequencing, or Southern blotting for further characterization.

1 Physical basis

2 Types of gel

2.1 Agarose

2.2 Polyacrylamide

2.3 Starch

3 Gel conditions

3.1 Denaturing

3.2 Native

4 Buffers

5 Visualization

6 Downstream processing

7 Applications

7.1 Nucleic acids

7.2 Proteins

8 History

9 See also

10 References

11 External links

Physical basis[edit]
See also: electrophoresis

In simple terms, electrophoresis is a process which enables the sorting of molecules based on size.
Using an electric field, molecules (such as DNA) can be made to move through a gel made
of agar or polyacrylamide. The electric field consists of a negative charge at one end which pushes
the molecules through the gel, and a positive charge at the other end that pulls the molecules
through the gel. The molecules being sorted are dispensed into a well in the gel material. The gel is
placed in an electrophoresis chamber, which is then connected to a power source. When the electric
current is applied, the larger molecules move more slowly through the gel while the smaller
molecules move faster. The different sized molecules form distinct bands on the gel. [citation needed]
The term "gel" in this instance refers to the matrix used to contain, then separate the target
molecules. In most cases, the gel is a crosslinked polymer whose composition and porosity is
chosen based on the specific weight and composition of the target to be analyzed. When
separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually
composed of different concentrations ofacrylamide and a cross-linker, producing different sized
mesh networks of polyacrylamide. When separating larger nucleic acids (greater than a few
hundred bases), the preferred matrix is purified agarose. In both cases, the gel forms a solid, yet
porous matrix. Acrylamide, in contrast to polyacrylamide, is a neurotoxin and must be handled using
appropriate safety precautions to avoid poisoning. Agarose is composed of long unbranched chains
of uncharged carbohydrate without cross links resulting in a gel with large pores allowing for the
separation of macromolecules and macromolecular complexes.[citation needed]
"Electrophoresis" refers to the electromotive force (EMF) that is used to move the molecules through
the gel matrix. By placing the molecules in wells in the gel and applying an electric field, the
molecules will move through the matrix at different rates, determined largely by their mass when the
charge to mass ratio (Z) of all species is uniform. However when charges are not all uniform then,
the electrical field generated by the electrophoresis procedure will affect the species that have
different charges and therefore will attract the species according to their charges being the opposite.
Species that are positively charged (cations) will migrate towards the cathode which is negatively
charged. If the species are negatively charged (anions) they will migrate towards the positively
charged anode.[4]
If several samples have been loaded into adjacent wells in the gel, they will run parallel in individual
lanes. Depending on the number of different molecules, each lane shows separation of the
components from the original mixture as one or more distinct bands, one band per component.
Incomplete separation of the components can lead to overlapping bands, or to indistinguishable
smears representing multiple unresolved components. [citation needed] Bands in different lanes that end up at
the same distance from the top contain molecules that passed through the gel with the same speed,
which usually means they are approximately the same size. There are molecular weight size
markersavailable that contain a mixture of molecules of known sizes. If such a marker was run on
one lane in the gel parallel to the unknown samples, the bands observed can be compared to those
of the unknown in order to determine their size. The distance a band travels is approximately
inversely proportional to the logarithm of the size of the molecule.[citation needed]
There are limits to electrophoretic techniques. Since passing current through a gel causes heating,
gels may melt during electrophoresis. Electrophoresis is performed in buffer solutions to reduce pH
changes due to the electric field, which is important because the charge of DNA and RNA depends

on pH, but running for too long can exhaust the buffering capacity of the solution. Further, different
preparations of genetic material may not migrate consistently with each other, for morphological or
other reasons.

Types of gel[edit]
The types of gel most typically used are agarose and polyacrylamide gels. Each type of gel is wellsuited to different types and sizes of analyte. Polyacrylamide gels are usually used for proteins, and
have very high resolving power for small fragments of DNA (5-500 bp). Agarose gels on the other
hand have lower resolving power for DNA but have greater range of separation, and are therefore
used for DNA fragments of usually 50-20,000 bp in size, but resolution of over 6 Mb is possible
with pulsed field gel electrophoresis(PFGE).[5] Polyacrylamide gels are run in a vertical configuration
while agarose gels are typically run horizontally in a submarine mode. They also differ in their
casting methodology, as agarose sets thermally, while polyacrylamide forms in a chemical
polymerization reaction.

Main article: Agarose gel electrophoresis
Agarose gels are made from the natural polysaccharide polymers extracted from seaweed. Agarose
gels are easily cast and handled compared to other matrices, because the gel setting is a physical
rather than chemical change. Samples are also easily recovered. After the experiment is finished,
the resulting gel can be stored in a plastic bag in a refrigerator.
Agarose gels do not have a uniform pore size, but are optimal for electrophoresis of proteins that are
larger than 200 kDa.[6] Agarose gel electrophoresis can also be used for the separation of DNA
fragments ranging from 50 base pair to several megabases (millions of bases), the largest of which
require specialized apparatus. The distance between DNA bands of different lengths is influenced by
the percent agarose in the gel, with higher percentages requiring longer run times, sometimes days.
Instead high percentage agarose gels should be run with a pulsed field electrophoresis (PFE),
or field inversion electrophoresis.
"Most agarose gels are made with between 0.7% (good separation or resolution of large 510kb
DNA fragments) and 2% (good resolution for small 0.21kb fragments) agarose dissolved in
electrophoresis buffer. Up to 3% can be used for separating very tiny fragments but a vertical
polyacrylamide gel is more appropriate in this case. Low percentage gels are very weak and may
break when you try to lift them. High percentage gels are often brittle and do not set evenly. 1% gels
are common for many applications."[7]

Main article: Polyacrylamide gel electrophoresis
Polyacrylamide gel electrophoresis (PAGE) is used for separating proteins ranging in size from 5 to
2,000 kDa due to the uniform pore size provided by the polyacrylamide gel. Pore size is controlled
by modulating the concentrations of acrylamide and bis-acrylamide powder used in creating a gel.

Care must be used when creating this type of gel, as acrylamide is a potent neurotoxin in its liquid
and powdered forms.
Traditional DNA sequencing techniques such as Maxam-Gilbert or Sanger methods used
polyacrylamide gels to separate DNA fragments differing by a single base-pair in length so the
sequence could be read. Most modern DNA separation methods now use agarose gels, except for
particularly small DNA fragments. It is currently most often used in the field of immunology and
protein analysis, often used to separate different proteins or isoforms of the same protein into
separate bands. These can be transferred onto anitrocellulose or PVDF membrane to be probed
with antibodies and corresponding markers, such as in a western blot.
Typically resolving gels are made in 6%, 8%, 10%, 12% or 15%. Stacking gel (5%) is poured on top
of the resolving gel and a gel comb (which forms the wells and defines the lanes where proteins,
sample buffer and ladders will be placed) is inserted. The percentage chosen depends on the size of
the protein that one wishes to identify or probe in the sample. The smaller the known weight, the
higher the percentage that should be used. Changes on the buffer system of the gel can help to
further resolve proteins of very small sizes.[8]

Partially hydrolysed potato starch makes for another non-toxic medium for protein electrophoresis.
The gels are slightly more opaque than acrylamide or agarose. Non-denatured proteins can be
separated according to charge and size. They are visualised using Napthal Black or Amido Black
staining. Typical starch gel concentrations are 5% to 10%.[9][10][11]

Gel conditions[edit]
TTGE profiles representing the bifidobacterial diversity of fecal samples from two healthy volunteers (A and B)
before and after AMC (Oral Amoxicillin-Clavulanic Acid) treatment

Denaturing gels are run under conditions that disrupt the natural structure of the analyte, causing it
to unfold into a linear chain. Thus, the mobility of each macromolecule depends only on its linear
length and its mass-to-charge ratio. Thus, the secondary, tertiary, and quaternary levels
of biomolecular structure are disrupted, leaving only the primary structure to be analyzed.
Nucleic acids are often denatured by including urea in the buffer, while proteins are denatured
using sodium dodecyl sulfate, usually as part of the SDS-PAGE process. For full denaturation of
proteins, it is also necessary to reduce the covalent disulfide bonds that stabilize
their tertiary and quaternary structure, a method called reducing PAGE. Reducing conditions are
usually maintained by the addition of beta-mercaptoethanol or dithiothreitol. For general analysis of
protein samples, reducing PAGE is the most common form of protein electrophoresis.
Denaturing conditions are necessary for proper estimation of molecular weight of RNA. RNA is able
to form more intramolecular interactions than DNA which may result in change of its electrophoretic

mobility. Urea,DMSO and glyoxal are the most often used denaturing agents to disrupt RNA
structure. Originally, highly toxicmethylmercury hydroxide was often used in denaturing RNA
electrophoresis,[12] but it may be method of choice for some samples.[13]
Denaturing gel electrophoresis is used in the DNA and RNA banding pattern-based
methods DGGE (denaturing gradient gel electrophoresis),[14] TGGE (temperature gradient gel
electrophoresis), and TTGE (temporal temperature gradient electrophoresis). [15]


Specific enzyme-linked staining:Glucose-6-Phosphate Dehydrogenaseisoenzymes in Plasmodium

falciparuminfected Red blood cells[16]

Native gels are run in non-denaturing conditions, so that the analyte's natural structure is
maintained. This allows the physical size of the folded or assembled complex to affect the mobility,
allowing for analysis of all four levels of the biomolecular structure. For biological samples,
detergents are used only to the extent that they are necessary to lyse lipid membranes in the cell.
Complexes remainfor the most partassociated and folded as they would be in the cell. One
downside, however, is that complexes may not separate cleanly or predictably, as it is difficult to
predict how the molecule's shape and size will affect its mobility.

Unlike denaturing methods, native gel electrophoresis does not use a charged denaturing agent.
The molecules being separated (usuallyproteins or nucleic acids) therefore differ not only
in molecular mass and intrinsic charge, but also the cross-sectional area, and thus experience
different electrophoretic forces dependent on the shape of the overall structure. For proteins, since
they remain in the native state they may be visualised not only by general protein staining reagents
but also by specific enzyme-linked staining.
Native gel electrophoresis is typically used in proteomics and metallomics. However, native PAGE is
also used to scan genes (DNA) for unknown mutations as in Single-strand conformation

Buffers in gel electrophoresis are used to provide ions that carry a current and to maintain the pH at
a relatively constant value. There are a number of buffers used for electrophoresis. The most
common being, for nucleic acids Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA(TBE). Many other
buffers have been proposed, e.g. lithium borate, which is almost never used, based on Pubmed
citations (LB), iso electric histidine, pK matched goods buffers, etc.; in most cases the purported
rationale is lower current (less heat) and or matched ion mobilities, which leads to longer buffer life.
Borate is problematic; Borate can polymerize, and/or interact with cis diols such as those found in
RNA. TAE has the lowest buffering capacity but provides the best resolution for larger DNA. This
means a lower voltage and more time, but a better product. LB is relatively new and is ineffective in
resolving fragments larger than 5 kbp; However, with its low conductivity, a much higher voltage
could be used (up to 35 V/cm), which means a shorter analysis time for routine electrophoresis. As
low as one base pair size difference could be resolved in 3% agarose gel with an extremely low
conductivity medium (1 mM Lithium borate).[17]
Most SDS-PAGE protein separations are performed using a "discontinuous" (or DISC) buffer
system that significantly enhances the sharpness of the bands within the gel. During electrophoresis
in a discontinuous gel system, an ion gradient is formed in the early stage of electrophoresis that
causes all of the proteins to focus into a single sharp band in a process called isotachophoresis.
Separation of the proteins by size is achieved in the lower, "resolving" region of the gel. The
resolving gel typically has a much smaller pore size, which leads to a sieving effect that now
determines the electrophoretic mobility of the proteins.

Further information: Gel electrophoresis of nucleic acids Visualization and Gel electrophoresis of
proteins Visualization
After the electrophoresis is complete, the molecules in the gel can be stained to make them visible.
DNA may be visualized using ethidium bromide which, when intercalated into
DNA, fluoresce under ultraviolet light, while protein may be visualised using silver
stain or Coomassie Brilliant Blue dye. Other methods may also be used to visualize the separation of
the mixture's components on the gel. If the molecules to be separated contain radioactivity, for

example in a DNA sequencing gel, an autoradiogram can be recorded of the gel. Photographs can
be taken of gels, often using a Gel Doc system.

Downstream processing[edit]
After separation, an additional separation method may then be used, such as isoelectric
focusing or SDS-PAGE. The gel will then be physically cut, and the protein complexes extracted
from each portion separately. Each extract may then be analysed, such as by peptide mass
fingerprinting or de novo peptide sequencing after in-gel digestion. This can provide a great deal of
information about the identities of the proteins in a complex.


Overview of Gel Electrophoresis.

Estimation of the size of DNA molecules following restriction enzyme digestion, e.g.
in restriction mapping of cloned DNA.
Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern

Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and biochemistry.
The results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging
device. The image is recorded with a computer operated camera, and the intensity of the band or
spot of interest is measured and compared against standard or markers loaded on the same gel.
The measurement and analysis are mostly done with specialized software.
Depending on the type of analysis being performed, other techniques are often implemented in
conjunction with the results of gel electrophoresis, providing a wide range of field-specific

Nucleic acids[edit]
Main article: Gel electrophoresis of nucleic acids

An agarose gel of a PCR product compared to a DNA ladder.

In the case of nucleic acids, the direction of migration, from negative to positive electrodes, is due to
the naturally occurring negative charge carried by their sugar-phosphate backbone.[18]
Double-stranded DNA fragments naturally behave as long rods, so their migration through the gel is
relative to their size or, for cyclic fragments, their radius of gyration. Circular DNA such as plasmids,
however, may show multiple bands, the speed of migration may depend on whether it is relaxed or
supercoiled. Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and
migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents
that disrupt the hydrogen bonds, such as sodium hydroxide or formamide, are used to denature the
nucleic acids and cause them to behave as long rods again.[19]
Gel electrophoresis of large DNA or RNA is usually done by agarose gel electrophoresis. See the
"Chain termination method" page for an example of a polyacrylamide DNA sequencing gel.
Characterization through ligand interaction of nucleic acids or fragments may be performed by
mobility shift affinity electrophoresis.
Electrophoresis of RNA samples can be used to check for genomic DNA contamination and also for
RNA degradation. RNA from eukaryotic organisms shows distinct bands of 28s and 18s rRNA, the
28s band being approximately twice as intense as the 18s band. Degraded RNA has less sharply
defined bands, has a smeared appearance, and intensity ratio is less than 2:1.

Main article: Gel electrophoresis of proteins

SDS-PAGE autoradiography The indicated proteins are present in different concentrations in the two

Proteins, unlike nucleic acids, can have varying charges and complex shapes, therefore they may
not migrate into the polyacrylamide gel at similar rates, or at all, when placing a negative to positive
EMF on the sample. Proteins therefore, are usually denatured in the presence of a detergent such
as sodium dodecyl sulfate (SDS) that coats the proteins with a negative charge.[3] Generally, the
amount of SDS bound is relative to the size of the protein (usually 1.4g SDS per gram of protein), so
that the resulting denatured proteins have an overall negative charge, and all the proteins have a
similar charge to mass ratio. Since denatured proteins act like long rods instead of having a complex
tertiary shape, the rate at which the resulting SDS coated proteins migrate in the gel is relative only
to its size and not its charge or shape.[3]
Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), by native gel electrophoresis, by quantitative preparative native continuous polyacrylamide
gel electrophoresis (QPNC-PAGE), or by 2-D electrophoresis.
Characterization through ligand interaction may be performed by electroblotting or by affinity
electrophoresis in agarose or by capillary electrophoresis as for estimation of binding constants and
determination of structural features like glycan content through lectin binding.

Countercurrent distribution





countercurrent distribution, in chemistry, a multistage solvent-extraction process,

one of many separation methods that can be employed in chemical analysis.
Substances are separated by this method on the basis of their different solubilities in
two immiscible liquids. These two liquids, flowing in opposite directions, are brought into
contact, mixed, and allowed to separate. The upper layer is transferred off in one
direction and the lower in another; this cycle of operations may be repeated as many
times as necessary to effect the desired separation.
A sample of a substance in contact with two solvents that do not dissolve in one another
seeks an equilibrium condition in which it is distributed between them; the ratio of the
concentrations in the two solvents, called the distribution coefficient, is characteristic of
the compound and of the solvent pair. Compounds that have dissimilar molecular
structures usually have widely different distribution coefficients, and mixtures of such
compounds can be separated satisfactorily by one or a few transfers between a suitable
solvent pair in simple equipment. Closely similar substances, however, such as
proteins, have very similar distribution coefficients, and hundreds of transfers may be
required to produce a complete separation.
The principle of countercurrent distribution is similar to that of chromatography; both
procedures are used for analysis and purification of mixtures of similar compounds.
Countercurrent Extraction - Craig Apparatus.
A method of multiple liquid-liquid extractions is countercurrent extraction, which permits the
separation of substances with different distribution coefficients (ratios). A clever design
known as Craig apparatus is used for this purpose (Lyman C. Craig, 1943).

Lyman C. Craig, 1906-1974 (Biography)

Craig apparatus consists of a series of glass tubes (r: 0, 1, 2..) that are designed and
arranged such that the lighter liquid phase is transferred from one tube to the next. The
liquid-liquid extractions are taking place simultaneously in all tubes of the apparatus which
is usually driven electromechanically. In the following animated picture of a single glass tube
the typical "extraction/transfer" cycle is shown.

The lower (heavier) phase of the two-phase solvent system (e.g. water, blue layer in the
picture) is the "stationary phase", whereas the upper (lighter) phase (e.g. hexane, red layer
in the picture) is the "mobile phase".
In the beginning, tube #0 contains the mixture of substances to be separated in the heavier
solvent and all the other tubes contain equal volumes of the same solvent. The lighter
solvent is added to tube #0, extraction (equilibration) takes place and the phases are
allowed to separate. The upper phase of tube #0 is then transferred to tube #1 and fresh
solvent is added to tube #0, and the phases are equilibrated again. The upper layers of
tubes #0 and #1 are simultaneously transferred to tubes #1 and #2 respectively. This cycle
is repeated to carry on the process through the other tubes of the apparatus. Obviously,
substances with higher distribution ratio move faster than those with a lower distribution
It is interesting to examine the distribution of a substance A in each tube after a given
number of equilibration/transfer cycles.
Supposing that the volumes of each solvent are equal (V), and let W represent the weight of
A in the sample, p and q represent the fraction of A with distribution ratio of D in the upper
(organic solvent, o) and lower (water, w) phase, then it is

Since p+q = 1, we have

The fractions of solute in successive tubes after each extraction step are shown in the
following figure:

We observe that after n transfers/equilibration cycles, and since the ratio D=p/q must be
maintained for each tube after the equilibration step, the total fraction of A in each tube
corresponds to the terms of the binomial expansion (p+q) n. Therefore the total fraction of
solute in tube r after n transfers is given by (remember that 0!=1).

By combining with the previous expressions of p and q we finally obtain

The greater the difference of the distribution ratio of various substances, the better the
separation between each other. A much larger number of tubes is required to separate
mixtures of substances with almost similar distribution ratios.
The Craig apparatus is only rarely used because modern chromatographic techniques are by
far more efficient and convenient. However countercurrent extraction is educationally very
useful since it introduces the student to the fundamental concept of equilibration between
mobile and static phases. Each tube where a complete equilibration takes place corresponds
to one theoretical plate of the chromatographic column. Craig apparatuses with more than
about 100 tubes are very difficult are very difficult to construct and operate, and they must
be compared with modern chromatographic columns that may exhibit efficiencies of tens of
thousands theoretical plates.

A manually operated Craig apparatus consisting of 25 tubes (from:, -> history).

This applet demonstrates the principles of countercurrent extraction. A mixture of two
substances: A (with green color) and B (with red color) is going to be separated using a Craig
apparatus consisting of 20 (0, 1, .. 19) equilibration tubes. The user must enter the values of
distribution ratios for the two substances in the corresponding edit boxes.
By clicking the button START the experiment starts with tube #0 containing the mixture in
the lower phase, pure (lighter) solvent in the upper phase and the other tubes (#1-#19)
containing pure (heavier) solvent).
By clicking the button Extract an equilibration takes place and the two substances are
distributed between the lower and the upper phase according to their distribution ratio.
Then, by clicking the button Transfer the upper phase of tube #0 is transfer to tube #1 and
fresh upper phase to tube #0. The cycle can be repeated as many times we wish.
The effectiveness of the separation (for the given values of distribution ratios) can be
observed by both the bargram indicating the fraction of each substance in each tube and
the intensity of the colors in the upper and lower phases in each tube.
Also, the eluted fraction (percent) of each substance from the last tube is shown.

Centrifugation and centrifuges

Centrifugation is a separation process which uses the action of centrifugal force to promote
accelerated settling of particles in a solid-liquid mixture. Two distinct major phases are formed in
the vessel during centrifugation :
The sediment
Usually does not have a uniform structure.
Find below an example of a sediment deposit :

The centrifugate or centrate which is the supernatant liquid.

Often clear though sometimes cloudy, due to the presence of very fine colloidal particles that are
not readily settled. However it may also contain several phases if the mixtures interstitial liquid
contains element with different densities, such as oils for example.


Centrifugal force
In a cylindrical vessel that rotates at an angular speed w(rad/s) or N (rpm) and contains a liquid
ring of mean radius R (m) the centrifugal acceleration Fc (m/s) to which the particles are subjected
Fc = w2R = 0.011 N2R
The forced exerted on a particle per unit of weight is expressed by:
Fc = 0.011 N2R (rs rL) x 1/g = G (rs rl),
with G = w2R / g = 0.11 N2R / 9.81 = 11.2 x 10-4 N2R
Where rs : density of particle
rl : density of interstitial liquid

Centrifuges achieve separation by means of the accelerated gravitational force achieved by a rapid

rotation. This can either replace normal gravity in the sedimentation of suspension or provide the
driving force in the filtration through a filter medium of some kind.
The most common application is separation of solid substances from high concentrated
suspensions. Used in this way for the treatment of sewage sludge it enables the dewatering with
the production of more or less consistent sediment depending on the nature of the sludge to be
treated, and the accelerated thickening of low concentration sludge.
The separation is similar in principle to that achieved in a gravity separation process. The driving
force is higher because is resulting from the rotation of the liquid: in the case of sedimentation,
where the driving force is resulting from the difference in density between the solids particles and
the liquid, the separation is achieved with a force from 1000 to 20000 times that of gravity.
Most centrifuges rotate thanks to some kind of motor drive. The types of centrifuge used for
sedimentation include:


tubular bowl

chamber bowl

imperforate basket

disk stack separator


Sedimenting centrifuges were invented for liquid solid separation and not for handling solids. It
soon became apparent that these machines had wider applications, which would involve the
presence of solid impurities, leading to use for separating solids from liquids.

Centrifugation Theory
Centrifugation is a process used to separate or concentrate
materials suspended in a liquid medium. The theoretical basis of this
technique is the effect of gravity on particles (including
macromolecules) in suspension. Two particles of different masses
will settle in a tube at different rates in response to gravity.
Centrifugal force (measured as xg, gravity) is used to increase this
settling rate in an instrument called a centrifuge. Two common
examples of the use of centrifugal force are: (1) When you do the
"around the world" trick with a yo-yo, it is centrifugal force that
makes the yo-yo body stay at the end of the string as you rotate it;

and (2) When you wash clothes in a washing machine, it is

centrifugal force generated in the "spin" cycle that forces water out
of the fabric to facilitate faster drying.
Centrifuges are devices used in a variety of scientific and technical
applications which spin carrier vessels (centrifuge tubes) at high
rotation speeds and very high centrifugal force. The centrifugal force
(expressed as # gravities or, # xg) generated is proportional to the
rotation rate of the rotor (in rpm) and the distance between the rotor
center and the centrifuge tube. Therefore, a given centrifuge may
use multiple rotor sizes to give flexibilty in choosing centrifugation
conditions. Each centrifuge has a special graph, a nomograph, or a
table which relates rotation rate (rpm) to centrifugal force (xg) for
each size of rotor it accepts.
Typically, the material to be "spun" is placed in a centrifuge tube
which is then placed in a rotor. The rotor is generally a dense metal
which dissipates heat quickly, and is of sufficient mass that it
generates momentum, i.e., once its spinning it requires little energy
to keep it going. Centrifuges generally work under vacuum and are
refrigerated to reduce heating caused by frictional forces as the
rotor spins. Rotors are usually stored in refrigeration units to keep
them at or near the operating temperature.
Centrifuges come in all shapes and sizes, and the rotors vary,
therefore, the universal and transferable unit of centrifugation is
centrifugal force in gravities (xg). Different makes of centrifuges use
different rotors and each model comes with a table or a graph that
relates centrifugal force to rotational speed (rpm) for each rotor (or
swing buckets) it can use. In lab write-ups you should ALWAYS report
the centrifugal force used (#gravities) and duration of time at that
force because centrifugal force is the only transferable unit between
different centrifuges.
Top of page

Differential Centrifugation
A commonly used technique for cell fractionation, called differential
centrifugation, is used to separate particles from a liquid medium or
to separate particles of different masses into separate fractions of

the supernatant. We will use this technique in a several ways in this

1. In the Bio 242 Amylase lab we will use centrifugation to pellet the
cellular debris and excess starch during the enzyme extract
preparation. The enzyme, which is soluble, will remain in the
supernatant. During the actual experiment, we will use
centrifugation to separate the enzyme (soluble) from its substrate
(insoluble amylose-azure) to stop the reaction.
2. In the molecular labs we will use centrifugation to promote a
chemical reaction by forcing small quantities of reactants together in
the bottom of microcentrifuge tubes. We will also use centrifugation
to prepare bacterial cells for transformation by alternately pelleting
them and then resuspending them with different chemical solutions.
3. In the Hill Reaction lab we will use a multi-step differential
centrifugation (Fig. 9-3) to isolate cell organelles (chloroplasts) from
crude cellular homogenate. Because the organelles have much less
mass than the cell wall components, the first pellet that forms at low
centrifugal force is primarily cellular debris. The organelle fraction is
then pelleted at higher centrifugal force.
Top of page

Centrifuge Cautions:
These cautions presume you have had proper instruction in the use
of the centrifuge AND have read the instructions for using the
instrument thoroughly.
1. Make sure the correct rotor is being used and that it is installed
properly on the spindle. Make sure the rotor is secured before
starting a run. On the prep centrifuges the rotor cap screws onto the
2. Balance the load in the rotor - every tube must have a
balance tube in the opposite slot with the same volume of fluid.
Imbalanced rotors can damage or destroy the machine, and, in
some instances kill people.

3. Make sure you are using the appropriate centrifuge tube for the
job - they can rupture at too high a speed. You may need special,
high density tubes for high force centrifugation.
4. Pre-cool the centrifuge and the rotor before use. Rotors should be
stored in a refrigerator when possible.
5. DO NOT attempt to override any safety features of the centrifuge.
6. NEVER leave the centrifuge unattended until it reaches maximum
speed and is going smoothly.
7. When in doubt, ASK FOR HELP.

9.4 Properties of Solutions

Cell walls are semipermeable membranes, so the osmotic pressures of the bodys fluids have
important biological consequences. If solutions of different osmolarity exist on either side of the
cells, solvent (water) may pass into or out of the cells, sometimes with disastrous results. Consider
what happens if red blood cells are placed in a hypotonic solution, meaning a solution of lower
osmolarity than the liquid inside the cells. The cells swell up as water enters them, disrupting cellular
activity and eventually causing the cells to burst. This process is called hemolysis. If red blood cells
are placed in a hypertonicsolution, meaning one having a higher osmolarity than exists inside the
cells, water leaves the cells to dilute the external solution, and the red blood cells shrivel and die.
This process is called crenation. Only if red blood cells are placed in isotonic solutions that have the
same osmolarity as exists inside the cells are they unaffected by negative effects of osmotic pressure.
Glucose solutions of about 0.31 M, or sodium chloride solutions of about 0.16 M, are isotonic with
blood plasma.

Cell Lysis Solutions

Cell lysis solutions are detergent-based, buffers and reagent sets that have
been optimized for particular cell lysis applications. Effective cell lysis and

protein extraction for different species of organisms and different cell and
tissue types require different buffer formulations. This article briefly
describes available reagents for whole cell lysis and protein extraction. For a
discussion of cell fractionation and organelle isolation, see the link in the

Learn more

Overview of cell lysis

Traditional cell lysis methods

Detergents for cell lysis

Cell lysis using detergents

Detergent cell lysis is a milder and easier alternative to physical disruption of cell membranes, although it
is often used in conjunction with homogenization and mechanical grinding. Detergents break the lipid
barrier surrounding cells by solubilizing proteins and disrupting lipid:lipid, protein:protein and protein:lipid
interactions. Detergents, like lipids, self associate and bind to hydrophobic surfaces. They are comprised
of a polar hydrophilic head group and a nonpolar hydrophobic tail and are categorized by the nature of the
head group as either ionic (cationic or anionic), nonionic or zwitterionic. Their behavior depends on the
properties of the head group and tail.
Unfortunately, there is no standard protocol available for selecting a detergent to use for membrane lysis.
In general, nonionic and zwitterionic detergents are milder and less denaturing than ionic detergents and
are used to solubilize membrane proteins where it is critical to maintain protein function and/or retain
native protein:protein interactions for enzyme assays or immunoassays. CHAPS, a zwitterionic detergent,
and the Triton-X series of nonionic detergents are commonly used for these purposes. In contrast, ionic
detergents are strong solubilizing agents and tend to denature proteins, thereby destroying protein activity
and function.
The choice of detergent for cell lysis also depends on sample type. Animal cells, bacteria and yeast all
have differing requirements for optimal lysis due to the presence or absence of a cell wall. Because of the
dense and complex nature of animal tissues, they require both detergent and mechanical lysis. In addition
to the choice of detergent, other important considerations for optimal cell lysis include the buffer, pH, salt
concentration and temperature. Consideration should be given to the compatibility of the chosen

detergent with downstream applications. If the detergent used for lysis must be removed, then a
dialyzable detergent should be selected.]


When animal cells are placed in a hypotonic solution, they will take
inwater due to osmosis. If the solution in which they are placed is a low enough
concentration, such as distilled water, the intake of water will make the cells swell up
and eventually burst.
What Happens to an Animal Cell in a Hypotonic Solution?
by Kevin Carr, Demand Media

Red blood cells, found in many larger animals, react to higher and lower concentrations of solution in blood.

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Both plants and animals have cells, and one of the main differences between them is that
plant cells have a cell wall. This helps the cells retain their shape even if their environment
changes considerably. Animal cells are more flexible, and without the cell wall, they can
react more adversely to changes in their environment, such as the concentration of a
solution around them.

Plasma Membrane
All cells are surrounded by a plasma membrane, which is a double layer of phospholipids
that regulate the transport of materials into and out of the cell. In a plant, the cell
membrane is next to the cell wall, which helps give it structure. The plasma membrane of a
cell is said to be differentially permeable because it allows certain substances, such as
water, to flow freely through it, but not others, such as ions and large molecules.

Diffusion and Osmosis

A solution contains the solvent, the liquid, such as water and the solute, the solids dissolved
into the solvent. In a solution, particles in an area of high concentration will naturally move
to an area of low concentration. This is known as diffusion. When diffusion happens across a
differentially permeable membrane, it is known as osmosis. This is important to cells
because if the solute cannot move across the differentially permeable membrane, water will
freely move into or out of a cell to balance out the concentration.

Isotonic Solution
If a cell is in a solution that has the same concentration outside the cell as it is inside the
cell, it is said to be isotonic. This is the ideal situation for a cell, and the flow of water into
the cell equals the flow of water out of the cell. However, cells often encounter environments
that have different concentrations of solution, such as organisms found in fresh water (low
concentration of solute) or ocean water (high concentration of solute).

Hypotonic Solution
If the solution surrounding a cell is less concentrated than that inside the cell, it is said to be
hypotonic. In a hypotonic solution, water will move from outside to the inside of the cell
across its membrane. This process will continue until the solutions reach equal
concentration, the cell ruptures from internal pressure or organelles inside the cell exert
energy to pump water out of the cell.

Hypertonic Solution
If a cell is in a solution that has a higher concentration than what is inside the cell, it is said
to be hypertonic. When this happens, water will flow out of the cell and into the solution.
This will continue until the concentrations are balanced, the cell dehydrates and collapses or
the organelles in the cell exert energy to pump water back into the cell.

Animal Cells vs. Plant Cells

When animal cells are placed in a hypotonic solution, they will take in water due to osmosis.
If the solution in which they are placed is a low enough concentration, such as distilled
water, the intake of water will make the cells swell up and eventually burst. This can, of
course, be a very dangerous result for cells placed in a solution of low concentration. Plant
cells do not rupture because the rigid cell walls hold them together.

Is distilled water hypertonic or



With regard to osmosis, distilled water will always be hypotonic compared to an

aqueous solution containing any amount of a solute.Because distilled water is pure
and contains no dissolved substances, an aqueous solution with any concentration of
solute will be hypertonic when compared to distilled water. Osmosis is a process based
on the concentration of solute contained in two aqueous solutions on either side of a
semipermeable membrane, and is not dependent on the dissolved substance.


What is the difference between distilled water and tap water?

How does water move during osmosis?

What is a hydration sphere?


When aqueous solutions are separated by a semipermeable membrane, the solution

containing the lesser concentration of solute, the hypotonic solution, will pass over to
the side containing the greater concentration of solute, which is the hypertonic solution.
Only the water will pass through the membrane, leaving the solute behind.
Osmotic pressure refers to the diffusion of water across a semipermeable membrane,
and is the result of two aqueous solutions striving toward a state of equilibrium. When
this state is reached, the osmotic pressure is the same on both sides. This equalized
degree of osmotic pressure is called the hydrostatic, or "water-stopping," pressure.
Because plant cells contain enzymes, salts and proteins dissolved in an aqueous
solution, a simple school lab experiment can be used to demonstrate osmotic pressure.
Placing a stalk of celery in a beaker of distilled water will cause the hypotonic water in
the beaker to flow across the celery stalk's cell membranes to equalize the pressure
difference. The celery stalk will become rigid as its cells fill with distilled water. Placing
another celery stalk in a beaker containing a salt solution will cause the reverse effect.
The water leaving the hypotonic solution within the plant cells to cross over to the
hypertonic salt solution in the beaker will cause the celery stalk to shrivel and become
DNA is considered denatured when the double stranded DNA molecule is converted
into ... G+C content of a DNA molecule can be estimated from its thermal melting ...The more
dense the DNA the quicker it will sediment upon centrifugation.

Section 3.5Purifying, Detecting, and Characterizing Proteins

A protein must be purified before its structure and the mechanism of its action can be studied.
However, because proteins vary in size, charge, and water solubility, no single method can be
used to isolate all proteins. To isolate one particular protein from the estimated 10,000 different
proteins in a cell is a daunting task that requires methods both for separating proteins and for
detecting the presence of specific proteins.

Any molecule, whether protein, carbohydrate, or nucleic acid, can be separated from other
molecules based on large differences in some physical characteristic. Although the sequence of
amino acids in a protein uniquely determines its function, the most useful physical characteristic
for separation of proteins is size, defined as either length or mass. In this section, we briefly
outline different techniques for separating proteins based on their size and other properties.
These techniques also apply to the separation of nucleic acids and other biomolecules. We then
consider general methods for detecting, or assaying, specific proteins, including the use of
radioactive compounds for tracking biological activity. Finally, we discuss several techniques for
characterizing a proteins mass, sequence, and three-dimensional structure.
Go to:

Proteins Can Be Removed from Membranes by Detergents or High-Salt Solutions

Because water-soluble globular proteins have many exposed hydrophilic groups, they maintain
their nativeconformation and remain individually suspended in an aqueous medium when
separated from cells. In contrast, when transmembrane proteins are separated from membranes,
their exposed hydrophobic regions interact, causing the proteinmolecules to aggregate and
precipitate from aqueous solutions. Such proteins can be solubilized by detergents, which have
affinity both for hydrophobic groups and for water.
Detergents are amphipathic molecules that disrupt membranes by intercalating into phospholipid
bilayers and solubilizing lipids and proteins. The hydrophobic part of a detergent molecule is
attracted to hydrocarbons and mingles with them readily; the hydrophilic part is strongly
attracted to water. Some detergents are natural products, but most are synthetic molecules
developed for cleaning and for dispersing mixtures of oil and water (Figure 3-38). Ionic
detergents, such as sodium deoxycholate and sodium dodecylsulfate (SDS), contain a charged
group; nonionic detergents, such as Triton X-100 and octylglucoside, lack a charged group.

Figure 3-38

Structures of five common detergents. The bile salt sodium deoxycholate is a natural product; the
others are synthetic ones. The hydrophobic portion of each molecule is shown in yellow; the
hydrophilic portion, in blue.

At very low concentrations, detergents dissolve in pure water as isolated molecules. As the
concentration increases, the molecules begin to form micelles. These are small, spherical
aggregates in which hydrophilic parts of the molecules face outward and the hydrophobic parts
cluster in the center (see Figure 2-20). The critical micelle concentration (CMC) at which
micelles form is characteristic of each detergent and is a function of the structures of its
hydrophobic and hydrophilic parts.
Ionic detergents bind to the exposed hydrophobic regions of membrane proteins as well as to the
hydrophobic core of water-soluble proteins. Because of their charge, these detergents also disrupt
ionic and hydrogen bonds. At high concentrations, for example, sodium dodecylsulfate
completely denatures proteins by binding to every side chain.Nonionic detergents act in different
ways at different concentrations. At high concentrations (above the CMC), they solubilize
biological membranes by forming mixed micelles of detergent, phospholipid, and integral
membrane proteins (Figure 3-39). At low concentrations (below the CMC), these detergents may
bind to the hydrophobic regions of most membrane proteins, making them soluble in aqueous
solution. In this case, although mixed micelles are not formed, the solubilized protein will not
aggregate during subsequent purification steps.

Figure 3-39

Solubilization of integral membrane proteins by nonionic detergents. At a concentration higher

than its critical micelle concentration (CMC), a detergent solubilizes lipids and integral
membrane proteins, forming mixed micelles containing (more...)
Most peripheral proteins are bound to specific integral membrane proteins by ionic or other weak
interactions. Generally they can be removed from the membrane by solutions of high ionic
strength (high salt concentrations), which disrupt ionic bonds, or by chemicals that bind divalent
cations such as Mg2+. Unlike integral proteins most peripheral proteins are soluble in aqueous
solution and are not solubilized by nonionic detergents.
Go to:

Centrifugation Can Separate Particles and Molecules That Differ in Mass or Density
The first step in a typical protein-purification scheme is centrifugation. The principle behind
centrifugation is that two particles in suspension (cells, organelles, or molecules), having

different masses or densities will settle to the bottom of a tube at different rates.
Remember, mass is the weight of a sample (measured in grams), whereas density is the ratio of
its weight to volume (grams/liter). Proteins vary greatly in mass but not in density. The average
density of a protein is 1.37 g/cm3. Unless a protein has an attached lipid or carbohydrate, its
density will not vary by more than 15 percent from this value. Table 3-1 lists the density and
other physical characteristics of several blood proteins. Heavier or more dense molecules settle,
or sediment, more quickly than lighter or less dense molecules.

Table 3-1

Physical Characteristics of Selected Blood Proteins.

A centrifuge speeds sedimentation by subjecting particles in suspension to centrifugal forces as
great as 600,000 times the force of gravity g. The centrifugal force is proportional to the rotation
rate of the rotor (measured in revolutions per minute, or rpm) and the distance of the tube from
the center of the rotor. Modern ultracentrifuges reach speeds of 60,000 rpm or greater and
generate forces sufficient to sediment particles with masses greater than 10,000 daltons (Da).
However, small particles with masses of 5 Da or less will not sediment uniformly even at such
high rotor speeds.
Centrifugation is used for two basic purposes: (1) as a preparative technique to separate one type
of material from others and (2) as an analytical technique to measure physical properties (e.g.,
molecular weight, density, shape, and equilibrium binding constants) of macromolecules.
The sedimentation constant, s, of a protein equals its velocity in a centrifugal field divided by the
centrifugal force. The value of s depends on a proteins density and shape, as well as the density
and viscosity of the medium. Because the centrifugal force and the density and viscosity of the
medium are all known, the radius and mass of a molecule can be calculated from measurements
of its rate of movement in an analytical ultracentrifuge. The sedimentation constant is commonly
expressed in svedbergs (S): 1 S=1013 seconds.

Differential Centrifugation

The most common initial step in protein purification is separation of soluble proteins from
insoluble cellular material bydifferential centrifugation (Figure 3-40a). The centrifugal force and
duration of centrifugation are adjusted to ensure that the insoluble materials sediment into a
pellet. After a starting mixture of a cell homogenate is poured into a tube and spun in a
centrifuge, cell organelles such as nuclei collect into a pellet, but the soluble proteins remain in
the supernatant. The supernatant fraction still contains a large mixture of proteins, which can be
collected by decanting the supernatant and then subjecting it to further purification methods.

Figure 3-40

Two common centrifugation techniques for separating particles. (a) Differential centrifugation
separates a mixture of particles (macromolecules, cell organ-elles, and cells) that differ in mass
or density. The most dense particles collect at(more...)
Rate-Zonal Centrifugation

Based on differences in their mass, proteins can be separated by centrifugation through a

solution, usually containing sucrose (an inert sugar), of increasing density called a density
gradient. When mixtures of proteins are layered on top of a sucrose gradient in a tube and
subjected to centrifugation, they migrate down the tube at a rate controlled by the factors that
affect the sedimentation constant. The proteins start from a thin zone at the top of the tube and
separate into bands, or zones (actually disks), of proteins of different masses. This densitygradient separation technique is called rate-zonal centrifugation (Figure 3-40b). Samples are
centrifuged just long enough to separate the molecules of interest. If they are centrifuged for too
short a time, the molecules will not separate sufficiently. If they are centrifuged much longer than
necessary, all the molecules will end up in a pellet at the bottom of the tube.
Although the sedimentation rate is strongly influenced by particle mass, rate-zonal centrifugation
is seldom effective in determining precise molecular weights because variations in shape also
affect sedimentation rate. The exact effects of shape are hard to assess, especially for proteins

and single-stranded nucleic acid molecules that can assume many complex shapes. Nevertheless,
rate-zonal centrifugation has proved to be the most practical method for separating many
different types of polymers and particles. A second density-gradient technique,
called equilibrium density-gradient centrifugation, is used mainly to separate DNA or organelles
(see Figure 5-24).
Go to:

Electrophoresis Separates Molecules according to Their Charge:Mass Ratio

Electrophoresis is a technique for separating, or resolving, molecules in a mixture under the
influence of an applied electric field. Dissolved molecules in an electric field move, or migrate,
at a speed determined by their charge:mass ratio. For example, if two molecules have the same
mass and shape, the one with the greater net charge will move faster toward an electrode. The
separation of small molecules, such as amino acids and nucleotides, is one of the many uses
ofelectrophoresis. In this case, a small drop of sample is deposited on a strip of filter paper or
other porous substrate, which is then soaked with a conducting solution. When an electric field is
applied at the ends of the strip, small molecules dissolved in the conducting solution move along
the strip at a rate corresponding to the magnitude of their charge.
SDS-Polyacrylamide Gel Electrophoresis

Because many proteins or nucleic acids that differ in size and shape have nearly identical
charge:mass ratios,electrophoresis of these macromolecules in solution results in little or no
separation of molecules of different lengths. However, successful separation of proteins and
nucleic acids can be accomplished by electrophoresis in various gels(semisolid suspensions in
water) rather than in a liquid solution. Electrophoretic separation of proteins is most commonly
performed in polyacrylamide gels. These gels are cast between a pair of glass plates by
polymerizing a solution of acrylamide monomers into polyacrylamide chains and simultaneously
cross-linking the chains into a semisolid matrix. The pore size of a gel can be varied by adjusting
the concentrations of polyacrylamide and the cross-linking reagent.
When a mixture of proteins is applied to a gel and an electric current applied, smaller proteins
migrate faster than larger proteins through the gel. The rate of movement is influenced by the
gels pore size and the strength of the electric field. The pores in a highly cross-linked
polyacrylamide gel are quite small. Such a gel could resolve small proteins and peptides, but
large proteins would not be able to move through it.
In what is probably the most powerful technique for resolving protein mixtures, proteins are
exposed to the ionic detergent SDS (sodium dodecylsulfate) before and during

gel electrophoresis (Figure 3-41). SDS denatures proteins, causing multimeric proteins to
dissociate into their subunits, and all polypeptide chains are forced into extended conformations
with similar charge:mass ratios. SDS treatment thus eliminates the effect of differences in shape,
so that chain length, which reflects mass, is the sole determinant of the migration rate of proteins
in SDS-polyacrylamide electrophoresis. Even chains that differ in molecular weight by less than
10 percent can be separated by this technique. Moreover, the molecular weight of a protein can
be estimated by comparing the distance it migrates through a gel with the distances that proteins
of known molecular weight migrate.

Figure 3-41

SDS-polyacrylamide gel electrophoresis, a common technique for separating proteins at good

resolution. The protein mixture first is treated with SDS, a negatively charged detergent that
binds to proteins. This binding dissociates multimeric (more...)
Two-Dimensional Gel Electrophoresis

Electrophoresis of all cellular proteins through an SDS gel can separate proteins having
relatively large differences in molecular weight but cannot resolve proteins having similar
molecular weights (e.g., a 41-kDa protein from a 42-kDa protein). To separate proteins of similar
mass, another physical characteristic must be exploited. Most commonly, this is electric charge,
which is determined by the number of acidic and basic residues in a protein. Two unrelated
proteins having similar masses are unlikely to have identical net charges because their sequences,
and thus the number of acidand basic residues, are different.
In two-dimensional electrophoresis, proteins are separated in two sequential steps: first by their
charge and then by their mass. In the first step, a cell extract is fully denatured by high
concentrations (8 M) of urea and then layered on a glass tube filled with polyacrylamide that is
saturated with a solution of ampholytes, a mixture of polyanionic and polycationic molecules.
When placed in an electric field, the ampholytes will separate and form a continuous gradient
based on their net charge (Figure 3-42a). The most highly polyanionic ampholytes will collect at
one end of the tube, and the most polycationic ampholytes will collect at the other end. This

gradient of ampholytes establishes a pH gradient. Charged proteins will migrate through the
gradient until they reach their pI, or isoelectric point, the pH at which the net charge of
the protein is zero. This technique, called isoelectric focusing (IEF), can resolve proteins that
differ by only one charge unit.

Figure 3-42

Two-dimensional gel electrophoresis, a technique for separating proteins based on their charge
and their mass. (a) Preparation of a two-dimensional protein gel by isoelectric focusing (IEF)
followed by SDS electrophoresis. (b) Two-dimensional (more...)
Proteins that have been separated on an IEF gel can then be separated in a second dimension
based on their molecular weights. To accomplish this, the IEF gel is extruded from the tube and
placed lengthwise on a second polyacrylamide gel, this time formed as a slab saturated with
SDS. When an electric field is imposed, the proteins will migrate from the IEF gel into the SDS
slab gel and then separate according to their mass. The sequential resolution of proteins by their
charge and mass can achieve excellent separation of cellular proteins (Figure 3-42b). For
example, two-dimensional gels have been very useful in studying the expression of various genes
in differentiated cells because as many as 1000 proteins can be resolved simultaneously.
Go to:

Liquid Chromatography Resolves Proteins by Mass, Charge, or Binding Affinity

Liquid chromatography, a third commonly used technique to separate mixtures of proteins,
nucleic acids, and other molecules, is based on the principle that molecules dissolved in a
solution will interact (bind and dissociate) with a solid surface. If the solution is allowed to flow
across the surface, then molecules that interact frequently with the surface will spend more time
bound to the surface and thus move more slowly than molecules that interact infrequently with
the surface. Liquid chromatography is performed in a column packed tightly with spherical
beads. The nature of these beads determines whether separation of proteins depends on
differences in mass, charge, or binding affinity.

Gel Filtration Chromatography

Proteins that differ in mass can be separated by gel filtration chromatography. In this technique,
the column is composed of porous beads made from polyacrylamide, dextran (a
bacterial polysaccharide), or agarose (a seaweed derivative). Proteins flow around the spherical
beads in gel filtration chromatography. However, the surface of the beads is punctured by large
holes, and proteins will spend some time within these holes. Because smaller proteins can
penetrate into the beads more easily than larger proteins, they travel through a gel filtration
column more slowly than larger proteins (Figure 3-43a). (In contrast, proteins
migrate through the pores in an electrophoretic gel; thus smaller proteins move faster than larger
ones.) The total volume of liquid required to elute a protein from the column depends on its
mass: the smaller the mass, the greater the elution volume. By use of proteins of known mass, the
elution volume can be used to estimate the mass of a protein in a mixture.

Figure 3-43

Three commonly used liquid chromatographic techniques. (a) Gel filtration chromatography
separates proteins that differ in size. A mixture of proteins is carefully layered on the top of a
glass cylinder packed with porous beads. Smaller (more...)
Ion-Exchange Chromatography

In a second type of liquid chromatography, called ion-exchange chromatography, proteins are

separated based on differences in their charge. This technique makes use of specially modified
beads whose surfaces are covered by amino groups or carboxyl groups and thus carry either a
positive charge (NH3+) or a negative charge (COO) at neutral pH.
The proteins in a mixture carry various net charges at any given pH. When a solution of
a protein mixture flows through a column of positively charged beads, only proteins with a net
negative charge (acidic proteins) adhere to the beads; neutral and basic proteins flow unimpeded
through the column (Figure 3-43b). The acidic proteins are then eluted selectively by passing a
gradient of increasing concentrations of salt through the column. At low salt concentrations,
protein molecules and beads are attracted by their opposite charges. At higher salt concentrations,
negative salt ions bind to the positively charged beads, displacing the negatively charged
proteins. In a gradient of increasing salt concentration, weakly charged proteins are eluted first

and highly charged proteins are eluted last. Similarly, a negatively charged column can be used
to retain and fractionate positively charged (basic) proteins.
Affinity Chromatography

A third form of chromatography, called affinity chromatography, relies on the ability of

a protein to bind specifically to another molecule. Columns are packed with beads to which are
covalently attached ligand molecules that bind to the protein of interest. Ligands can
be enzyme substrates or other small molecules that bind to specific proteins. In a widely used
form of this technique, antibody-affinity chromatography, the attached ligand is an antibody
specific for the desired protein (Figure 3-43c). An affinity column will retain only the proteins
that bind the ligand attached to the beads; the remaining proteins, regardless of their charge or
mass, will pass through the column without binding to it. The proteins bound to the affinity
column then are eluted by adding an excess of ligand or by changing the salt concentration
or pH. Obviously, the ability of this technique to separate particular proteins depends on the
selection of appropriate ligands.
Go to:

Highly Specific Enzyme and Antibody Assays Can Detect Individual Proteins
Purification of a protein, or any other molecule, requires a specific assay that can detect the
molecule of interest in column fractions or gel bands. An assay capitalizes on some highly
distinctive characteristic of a protein: the ability to bind a particular ligand, to catalyze a
particular reaction, or to be recognized by a specific antibody. An assay must also be simple and
fast in order to minimize errors and the possibility that the protein of interest is denatured or
degraded while the assay is performed. The goal of any purification scheme is to isolate
sufficient amounts of a given protein for study; thus a useful assay must also be sensitive enough
that only a small proportion of the available material is consumed. Many common protein assays
require just 109 to 1012 g of material.
Chromogenic and Light-Emitting Enzyme Reactions

Many assays are tailored to detect some functional aspect of a protein. For
example, enzyme assays are based on the ability to detect the loss of substrate or the formation of
product. Many enzyme assays utilize chromogenic substrates, which change color during the
course of the reaction. (Some substrates are naturally chromogenic; if they are not, they can be
linked to a chromogenic molecule.) Because of the specificity of an enzyme for its substrate,
only samples that contain the enzyme will change color in the presence of a chromogenic

substrate and other required reaction components; the rate of the reaction provides a measure of
the quantity of enzyme present.
Such chromogenic enzymes also can be fused or chemically linked to an antibody and used to
report the presence or location of the antigen. Alternatively luciferase, an enzyme present in
fireflies and some bacteria, can be linked to an antibody. In the presence of ATP and luciferin,
this enzyme catalyzes a light-emitting reaction. In either case, after the antibody binds to
the protein of interest, substrates of the linked enzyme are added and the appearance of color or
emitted light is monitored.
Western Blotting

One of the most powerful methods for detecting a particular protein in a complex mixture
combines the superior resolving power of gel electrophoresis, the specificity of antibodies, and
the sensitivity of enzyme assays. CalledWestern blotting, or immunoblotting, this three-step
procedure is commonly used to separate proteins and then identify a specific protein of interest.
As shown in Figure 3-44, two different antibodies are used in this method, one specific for the
desired protein and the other linked to a reporter enzyme.

Figure 3-44

Western blotting, or immunoblotting. (a) A protein mixture is electrophoresed through an SDS

gel, and then transferred from the gel onto a membrane. (b) The membrane is flooded with a
solution of antibody (Ab1) specific for the desired(more...)
Go to:

Radioisotopes Are Indispensable Tools for Detecting Biological Molecules

Since World War II, when radioactive materials first became widely available as byproducts of
work in nuclear physics, chemists and biologists have fashioned an almost limitless variety of
radioactive chemicals. Today, radioactively labeled precursors of macromolecules greatly
simplify many standard biochemical assays and significantly enhance the ability of researchers to
follow biochemical events in whole cells as well as in cell extracts. Almost all experimental
biology depends on the use of radioactive compounds.

At least one atom in a radiolabeled molecule is present in a radioactive form, called

a radioisotope (Table 3-2). The presence of a radioisotope does not change the chemical
properties of a molecule. For example, enzymes, both in vivoand in vitro, catalyze reactions
involving labeled substrates just as readily as those involving nonlabeled substrates. Because
radioisotopes emit easily detected particles, the fate of radiolabeled molecules can be traced in
cells and cellular extracts.

Table 3-2

Radioisotopes Commonly Used in Biological Research.

Characteristics of Different Radiolabels

The choice of which labeled compound to use in a particular experiment involves several
considerations. Some labeled compounds, for instance, are not suitable for studies with whole
cells because they do not enter cells. One prominent example is ATP, as well as most other
phosphorylated compounds (e.g., glucose 6-phosphate). Although 32P-labeled ATP can contribute
phosphorus-32 during RNA and DNA synthesis in a cell-free system, it cannot do so with whole
cells, because it never gets into the cells. On the other hand, labeled orthophosphate (32PO43) in
the medium does enter both bacterial cells and animal cells, and then is incorporated into
phosphorylated proteins, nucleotides, and eventually into cellular RNA and DNA.
Hundreds of biological compounds (e.g., amino acids, nucleosides, and numerous metabolic
intermediates) labeled with various radioisotopes are commercially available. These preparations
vary considerably in their specific activity, which is the amount of radioactivity per unit of
material, measured in disintegrations per minute (dpm) per millimole. The specific activity of a
labeled compound depends on the ratio of unstable potentially radioactive atoms to stable
nonradioactive atoms. It also depends on the probability of decay of the radioisotope, indicated
by its half-life, which is the time required for half the atoms to undergo radioactive decay. In
general, the shorter the half-life of a radioisotope, the higher its specific activity (see Table 3-2).
The specific activity of a labeled compound must be high enough so that sufficient radioactivity
is incorporated into cellular molecules to be accurately detected. For example, methionine and

cysteine labeled with sulfur-35 (35S) are widely used to label cellular proteins because
preparations of these amino acids with high specific activities (>1015dpm/mmol) are available.
Likewise, commercial preparations of 3H-labeled nucleic acid precursors have much higher
specific activities than the corresponding 14C-labeled preparations. In most experiments, the
former are preferable because they allow RNA or DNA to be adequately labeled after a shorter
time of incorporation or require a smaller cell sample.
Various phosphate-containing compounds in which every phosphorus atom is
the radioisotope phosphorus-32 are readily available. Because of their high specific activity, 32Plabeled nucleotides are routinely used to label nucleic acids in cell-free systems. The
radioisotope iodine-125 (125I), which also is available in almost pure form, can be covalently
linked to a protein or nucleic acid to yield preparations with a high specific activity. Such
attachment of iodine-125 can be achieved enzymatically or chemically and generally does not
drastically alter the properties of a macromolecule.
Labeling Experiments and Detection of Radiolabeled Molecules

Depending on the nature of an experiment, labeled compounds are detected by autoradiography,

a semiquantitative visual assay, or their radioactivity is measured in an appropriate counter, a
highly quantitative assay that can determine the concentration of a radiolabeled compound in a
sample. In some experiments, both types of detection are used.
In autoradiography, a cell or cell constituent is labeled with a radioactive compound and then
overlaid with a photographic emulsion sensitive to radiation. Development of the emulsion
yields small silver grains whose distribution corresponds to that of the radioactive material
(Figure 3-45a). Autoradiographic studies of whole cells have been crucial in determining the
intracellular sites where various macromolecules are synthesized and their subsequent
movements within cells. For example, when cells are incubated for a short time with
[3H]thymidine, a unique DNA precursor, most of the radioactivity is localized to the nucleus,
identifying the nucleus as the major site of DNA synthesis (Figure 3-45b). Even after cells are
incubated for prolonged periods with [3H]thymidine, virtually all the radioactivity remains in the
nucleus, indicating that the DNA remains there. Similarly, the site of synthesis of RNA is
revealed by incubating cells for 1 minute with [3H]uridine, a unique RNA precursor; in this case,
all the autoradiographic grains are found over the nucleus. After a longer period of incorporation,
however, many autoradiographic grains are located over the cytoplasm, indicating that RNA, in
contrast to DNA, is transported from the nucleus.

Figure 3-45

Autoradiography. (a) A radiation-sensitive photographic emulsion containing silver salts (AgBr)

is placed over tritium-labeled cells attached to a glass slide (for the light microscope) or to a
carbon-coated grid (for the electron microscope). (more...)
In autoradiographic studies, the ability of the experimenter to localize the site at which
the radioisotope is incorporated is affected by the energy of the particles emitted during
radioactive disintegrations. For example, the particles emitted by phosphorus-32 are so
energetic that the streaks they make on a photographic emulsion can be as long as 1 mm, much
longer than the diameter of individual cells. In contrast, the particles emitted by tritium create
tracks on a photographic emulsion that are only about 0.47 m long; thus 3H-labeled structures
can be located within cells to an accuracy of about 0.51.0 mm, or about one-fifth the diameter
of the nucleus of mammalian cells. Because tritium emits the least-energetic particles of all the
common radioisotopes, it is highly preferred for locating labeled compounds or structures within
Quantitative measurements of the amount of radioactivity in a labeled material are performed
with several different instruments. A Geiger counter measures ions produced in a gas by the
particles or rays emitted from a radioisotope. In a scintillation counter, a radiolabeled sample is
mixed with a liquid containing a fluorescent compound that emits a flash of light when it absorbs
the energy of the particles or rays released during decay of the radioisotope; a phototube in
the instrument detects and counts these light flashes. Phosphorimagers are used to detect
radiolabeled compounds on a surface, storing digital data on the number of decays in dpm per
small pixel of surface area. These instruments commonly are used to quantitate radioactive
molecules separated by gel electrophoresis and are replacing photographic film for this purpose.
The usual experimental protocol for determining the cellular location of a particular molecule
has three steps:

A radioactive precursor is incubated with whole cells or cell-free extracts.


The cellular constituents then are isolated and purified in various ways.

The radioactivity of the various fractions is measured with a counter.

For example, to identify the site of RNA synthesis, cells can be incubated for a short period with
[3H]uridine and then subjected to a fractionation procedure to separate the various organelles
(Chapter 5). The specific activity of the nuclear fraction (dpm/mg protein) is found to be much
higher than that of any other organelle fraction, thus confirming thenucleus as the site of RNA
A combination of labeling and biochemical techniques and of visual and quantitative detection
methods is often employed in labeling experiments. For instance, to identify the major proteins
synthesized by a particular cell type, a sample of the cells is incubated with a radioactive amino
acid (e.g., 35S-labeled methionine) for a few minutes. The mixture of cellular proteins then is
resolved by gel electrophoresis, and the gel is subjected to autoradiography or phosphorimager
analysis. The radioactive bands correspond to newly synthesized proteins, which have
incorporated the radiolabeled amino acid. Alternatively, the proteins can be resolved by liquid
chromatography, and the radioactivity in the eluted fractions determined quantitatively with a
Researchers often use the pulse-chase technique in labeling experiments. In this protocol, a cell
sample is exposed to a radiolabeled compound for only a brief period of time (the pulse), then
washed with buffer to remove the label, and finally incubated with a nonlabeled form of the
compound (the chase). Pulse-chase experiments are particularly useful for tracing changes in
the intracellular location of proteins or the transformation of a metabolite into others over time.
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Protein Primary Structure Can Be Determined by Chemical Methods and from Gene
The primary structure of a protein is characterized in two ways: by its overall amino
acid composition and by its precise amino acid sequence. The amino acid composition of a
protein gives the same information as an elemental analysis of a moleculethe types of amino
acids present and their abundance but not their linear order. In contrast, the sequence of a protein
is like a fingerprint; it uniquely establishes the identity of a proteinthe linear order of amino
acids. The composition of a protein is easily calculated from the sequence. Two proteins can
differ in their sequence but nonetheless have identical amino acid compositions. Composition
and sequence are determined by chemical methods based on the ability to cleave
the peptide backbone at the peptide bond.

The classic method for determining the amino acid sequence of a protein involves Edman
degradation (Figure 3-46). In this procedure the amino group at the N-terminus of
a polypeptide is labeled and its amino acid then cleaved from the polypeptide and identified by
high-pressure liquid chromatography. The polypeptide is left one residue shorter, with a new
amino acid at the N-terminus. The cycle is repeated on the ever shortening polypeptide until all
the residues have been identified.

Figure 3-46

Chemical determination of the sequence of a protein by Edman degradation, which involves a

repetitive three-step procedure. In the first step, the polypeptide Nterminus is reacted with
phenylisothiocyanate (PITC). In the second step, the (more...)
Before about 1985, biologists commonly used the Edman chemical procedure for
determining protein sequences. However, recombinant DNA techniques developed in the 1970s
and 1980s permit the detection and cloning of the mRNA or gene encoding a specific protein.
From the sequence of its mRNA or gene, the proteins amino acid sequence can be deduced.
Because sequencing of mRNA and DNA generally is faster than chemical sequencing of
proteins, this approach is now the most popular way to determine protein sequences, especially
of large proteins. As we describe in Chapter 7, the complete genome sequences of several
organisms have already been determined, and the database of genome sequences from humans
and numerous model organisms is expanding rapidly.
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Time-of-Flight Mass Spectrometry Measures the Mass of Proteins and Peptides

A powerful technique for measuring the mass of molecules like proteins and peptides is mass
spectrometry (Figure 3-47). Mass spectrometry requires a method for ionizing the sample,
usually a mixture of peptides or proteins, accelerating the molecular ions, and then detecting the
ions. In a laser desorption mass spectrometer, the proteins are mixed with an organic acid and
then dried on a metal target. Light from a laser ionizes the proteins, which fly down a tube to a

detector. Their time of flight is inversely proportional to their mass and directly proportional to
the charge on the protein. As little as 1 femtomole of proteins as large as 200,000 MW can be
measured with an error of 0.1 percent.

Figure 3-47

Mass measurements by time-of-flight mass spectrometry. Pulses of light from a laser ionize a
protein or peptide mixture that is absorbed on a metal target. An electric field accelerates the
molecules in the sample toward the detector.(more...)
One powerful use of mass spectrometers is to identify a protein from its peptide mass
fingerprint. A peptide mass fingerprint is a compilation of the molecular weights of peptides that
are generated by a specific protease. The molecular weights of the parent protein and its
proteolytic fragments are used to search genome databases for any similarly sized protein with
identical or similar peptide mass maps. With the increasing availability of genome sequences,
this approach has almost eliminated the need to chemically sequence a protein to determine
its primary structure.
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Peptides with a Defined Sequence Can Be Synthesized Chemically

Synthetic peptides that are identical with peptides synthesized in vivo are useful experimental
tools in studies of proteins and cells. For example, short synthetic peptides of 1015 residues can
function as antigens to trigger production of antibodies in animals. A synthetic peptide can trick
the animal into producing antibodies that bind the full-sized, naturalprotein antigen. As well see
throughout this book, antibodies are extremely versatile reagents for isolating proteins from
mixtures by affinity chromatography (see Figure 3-43c), separating and detecting proteins
by Western blotting (seeFigure 3-44), and localizing proteins in cells by microscopic techniques
described in Chapter 5. Synthetic peptides also have been helpful in elucidating the rules that
determine the secondary and tertiary structure of proteins. By systematically varying the
sequence of synthetic peptides, researchers have studied the influence of various amino acids on
protein conformation.
Peptides are routinely synthesized in a test tube from monomeric amino acids by condensation
reactions that formpeptide bonds. Peptides are constructed sequentially by coupling the Cterminus of a monomeric amino acid with the N-terminus of the growing peptide, as outlined

in Figure 3-48. To prevent unwanted reactions involving the amino groups and carboxyl groups
of the side chains during the coupling steps, a protecting (blocking) group is attached to the side
chains. Without these protecting groups, branched peptides would be generated. In the last steps
of synthesis, the side chainprotecting groups are removed and the peptide is cleaved from the
resin on which synthesis occurs.

Figure 3-48

Solid-phase peptide synthesis. The first amino acid (blue) of the desired peptide is attached at its
carboxyl end by esterification to a polystyrene bead. The amino group of this amino acid is
blocked by the attachment of a tertbutyloxycarbonyl(more...)
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Protein Conformation Is Determined by Sophisticated Physical Methods

In this chapter we have emphasized that protein function is derived from protein structure. Thus,
to figure out how a protein works, its three-dimensional structure must be known. Determining a
proteins conformation requires sophisticated physical methods and complex analyses of the
experimental data. We briefly describe three methods used to generate three-dimensional models
of proteins.
X-Ray Crystallography

The use of x-ray crystallography to determine the three-dimensional structures of proteins was
pioneered by Max Perutz and John Kendrew in the 1950s. To date, the detailed threedimensional structures of more than 8000 proteins have been established by this technique in
which beams of x-rays are passed through a crystal of protein. The wavelengths of x-rays are
about 0.10.2 nanometer (nm), short enough to resolve the atoms in the protein crystal. Atoms in
the protein crystal scatter the x-rays, which produce a diffraction pattern of discrete spots when

they are intercepted by photographic film (Figure 3-49). Such patterns are extremely complex; as
many as 25,000 diffraction spots can be obtained from a small protein. Elaborate calculations
and modifications of the protein (such as binding of heavy metals) must be made to interpret the
diffraction pattern and to solve the structure of the protein. The process is analogous to
reconstructing the precise shape of a rock from the ripples it creates in a pond.

Figure 3-49

X-ray diffraction. (a) Basic components of an x-ray crystallographic determination. When a

narrow beam of x-rays strikes a crystal, part of it passes straight through and the rest is scattered
(diffracted) in various directions. The(more...)
Cryoelectron Microscopy

Although some proteins readily crystallize, obtaining crystals of othersparticularly large

multisubunit proteinsrequires a time-consuming trial-and-error effort to find just the right
conditions. Low-resolution views of such difficult-to-crystallize proteins can be obtained by
electron microscopy (see Figure 4-34). In this technique, a protein sample is rapidly frozen in
liquid helium to preserve its structure and then examined in the frozen, hydrated state in
a cryoelectron microscope. Pictures are recorded on film using a low dose of electrons to prevent
radiation-induced damage to the structure. Sophisticated computer programs analyze the images
and reconstruct the proteins structure in three dimensions. With recent advances in cryoelectron
microscopy, this technique can generate molecular models that compare with those generated
by x-ray crystallography.
NMR Spectroscopy

The three-dimensional structures of small proteins containing up to about 200 amino acids can be
studied with nuclear magnetic resonance (NMR) spectroscopy. In this technique, a
concentrated protein solution is placed in a magnetic field and the effects of different radio
frequencies on the resonances of different atoms are measured. However, the behavior of any
atom is influenced by neighboring atoms in adjacent residues; the closely spaced residues are
more perturbed than distant residues. From the magnitude of the effect, the distances between
residues can be calculated; these distances then are used to generate a model of the threedimensional structure of the protein. Although NMR does not require crystallization of a protein,

a definite advantage, this technique is limited to proteins smaller than about 20 kDa. However,
NMR analysis also can be applied to protein domains, which tend to be small enough for this
technique and often can be obtained as stable structures.
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Proteins can be isolated based on differences in their physical and chemical

properties. Centrifugation,electrophoresis, and chromatography are the most
common techniques for purifying and analyzing proteins.

Centrifugation separates proteins based on their rate of sedimentation,

which is influenced by their mass and shape.

Gel electrophoresis separates proteins based on their rate of movement in an

applied electric field. SDSpolyacrylamide gel electrophoresis can
resolve polypeptide chains differing in molecular weight by 10 percent or less
(see Figure 3-41).

Liquid chromatography separates proteins based on their rate of movement

through a column packed with spherical beads. Proteins differing in mass are
resolved on gel filtration columns; those differing in charge, on ion-exchange
columns; and those differing in ligand-binding properties, on affinity columns
(see Figure 3-43).

Various assays are used to detect, identify, and quantify proteins. The most
sensitive assays use a lightproducing reaction or radioactivity to generate a
signal. Other assays produce an amplified colored signal with enzymes and
chromogenic substrates.

Antibodies are powerful reagents used to detect, quantify, and isolate

proteins. They are used in affinity chromatography and combined with
gel electrophoresis in Western blotting, a powerful method for separating and
detecting a protein in a mixture (see Figure 3-44).

Autoradiography is a semiquantitative technique for detecting radioactively

labeled molecules in cells, tissues, or electrophoretic gels (see Figure 3-45).

Three-dimensional structures of proteins are obtained by x-ray

crystallography, NMR spectroscopy, and cryoelectron microscopy. X-ray
crystallography provides the most detailed structures, but
requires proteincrystallization. Cryoelectron microscopy is particularly useful

for large protein complexes, which are difficult to crystallize. Only relatively
small proteins are amenable to NMR analysis.

Figure 3-38Structures of five common detergents

The bile salt sodium deoxycholate is a natural product; the others are
synthetic ones. The hydrophobic portion of each molecule is shown in
yellow; thehydrophilic portion, in blue.

From: Section 3.5, Purifying, De

Figure 3-39Solubilization of integral membrane proteins by nonionic detergents

At a concentration higher than its critical micelle concentration (CMC), a detergent

solubilizes lipids and integral membrane proteins, forming mixed micelles containing
detergent, protein, and lipid molecules. At concentrations below the CMC, many
detergents (e.g., octylglucoside) can dissolve membrane proteins without forming
micelles by coating the membrane-spanning regions. Since octylglucoside has a high
CMC, it is particularly effective in solubilizing integral membrane proteins without
denaturing them or forming mixed micelles.

Figure 3-40Two common centrifugation techniques for separating particles

(a) Differential centrifugation separates a mixture of particles (macromolecules, cell

organ-elles, and cells) that differ in mass or density. The most dense particles collect at
the bottom of the tube as a pellet. The least dense particles remain in the liquid
supernatant, which can be transferred to another tube. (b) Rate-zonal centrifugation
separates particles or molecules that differ in mass but may be similar in shape and
density (e.g., RNA molecules). Here two particles of different mass separate into two

Figure 3-40Two common centrifugation techniques for separating particles

(a) Differential centrifugation separates a mixture of particles (macromolecules, cell

organ-elles, and cells) that differ in mass or density. The most dense particles collect at
the bottom of the tube as a pellet. The least dense particles remain in the liquid
supernatant, which can be transferred to another tube. (b) Rate-zonal centrifugation
separates particles or molecules that differ in mass but may be similar in shape and
density (e.g., RNA molecules). Here two particles of different mass separate into two

Figure 3-41SDS-polyacrylamide gel electrophoresis, a common technique for

separating proteins at good resolution

The protein mixture first is treated with SDS, a negatively charged detergent that binds to
proteins. This binding dissociates multimeric proteins and forces all polypeptide chains
into dena-tured conformations with nearly identical charge:mass ratios.
During electrophoresis, the SDS-protein complexes migrate through the polyacrylamide

gel. Small proteins are able to move through the pores more easily, and faster, than larger
proteins. Thus the proteins separate into bands according to their size as they migrate
through the gel. The separated protein bands are visualized by staining with a dye.

Figure 3-42Two-dimensional gel electrophoresis, a technique for separating proteins

based on their charge and their mass

(a) Preparation of a two-dimensional protein gel by isoelectric focusing (IEF) followed

by SDS electrophoresis. (b) Two-dimensional gel of protein extracts from cells growing
in a medium. Each spot represents a single polypeptide; polypeptides can be detected by
dyes, as here, or by other techniques such as autoradiography. Each polypeptide is
characterized by its pI (isoelectric point) and molecular weight. [Part (b) courtesy of J.

Figure 3-43Three commonly used liquid chromatographic techniques

(a) Gel filtration chromatography separates proteins that differ in size. A mixture of
proteins is carefully layered on the top of a glass cylinder packed with porous beads.
Smaller proteins travel through the column more slowly than larger proteins. Thus
different proteins have different elution volumes and can be collected in separate liquid
fractions from the bottom. (b) Ion-exchange chromatography separates proteins that differ
in net charge in columns packed with special beads that carry either a positive charge
(shown here) or a negative charge. Proteins having the same net charge as the beads are

repelled and flow through the column, whereas proteins having the opposite charge bind
to the beads. Bound proteins, in this case negatively charged, are eluted by passing a salt
gradient (usually of NaCl or KCl) through the column. As the ions bind to the beads, they
desorb the protein. (c) In antibody-affinity chromatography, a specific antibody is
covalently attached to beads packed in a column. Only protein with high affinity for the
antibody is retained by the column; all the nonbinding proteins flow through. The bound
protein is eluted with an acidic solution, which disrupts the antigen-antibody complexes.

Section 4.1The Purification of Proteins Is an Essential First Step in

Understanding Their Function
An adage of biochemistry is, Never waste pure thoughts on an impure protein. Starting from pure
proteins, we can determine amino acid sequences and evolutionary relationships between
proteins in diverse organisms and we can investigate a protein's biochemical function. Moreover,
crystals of the protein may be grown from pure protein, and from such crystals we can obtain xray data that will provide us with a picture of the protein's tertiary structurethe
actual functional unit.
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4.1.1. The Assay: How Do We Recognize the Protein That We Are Looking For?
Purification should yield a sample of protein containing only one type of molecule, the protein in
which the biochemist is interested. This protein sample may be only a fraction of 1% of the
starting material, whether that starting material consists of cells in culture or a particular organ
from a plant or animal. How is the biochemist able to isolate a particular protein from a complex
mixture of proteins?
The biochemist needs a test, called an assay, for some unique identifying property of the protein
so that he or she can tell when the protein is present. Determining an effective assay is often
difficult; but the more specific the assay, the more effective the purification. For enzymes, which
are protein catalysts (Chapter 8), the assay is usually based on the reaction that the enzyme
catalyzes in the cell. Consider the enzyme lactate dehydrogenase, an important player in the
anaerobic generation of energy from glucose as well as in the synthesis of glucose from lactate.
Lactate dehydrogenase carries out the following reaction:

Nicotinamide adenine dinucleotide [reduced (NADH); Section 14.3.1] is distinguishable from

the other components of the reaction by its ability to absorb light at 340 nm. Consequently, we
can follow the progress of the reaction by examining how much light the reaction mixture
absorbs at 340 nm in unit timefor instance, within 1 minute after the addition of the enzyme.
Our assay for enzyme activity during the purification of lactate dehydrogenase is thus the
increase in absorbance of light at 340 nm observed in 1 minute.
To be certain that our purification scheme is working, we need one additional piece of
informationthe amount of protein present in the mixture being assayed. There are various rapid
and accurate means of determining protein concentration. With these two experimentally
determined numbersenzyme activity and protein concentrationwe then calculate the specific
activity, the ratio of enzyme activity to the amount of protein in the enzyme assay. The specific
activity will rise as the purification proceeds and the protein mixture being assayed consists to a
greater and greater extent of lactate dehydrogenase. In essence, the point of the purification is to
maximize the specific activity.
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4.1.2. Proteins Must Be Released from the Cell to Be Purified

Having found an assay and chosen a source of protein, we must now fractionate the cell into
components and determine which component is enriched in the protein of interest. Such
fractionation schemes are developed by trial and error, on the basis of previous experience. In the
first step, a homogenate is formed by disrupting the cell membrane, and the mixture is
fractionated by centrifugation, yielding a dense pellet of heavy material at the bottom of the
centrifuge tube and a lighter supernatant above (Figure 4.1). The supernatant is again centrifuged
at a greater force to yield yet another pellet and supernatant. The procedure, called differential
centrifugation, yields several fractions of decreasing density, each still containing hundreds of
different proteins, which are subsequently assayed for the activity being purified. Usually, one
fraction will be enriched for such activity, and it then serves as the source of material to which
more discriminating purification techniques are applied.

Figure 4.1

Differential Centrifugation. Cells are disrupted in a homogenizer and the resulting mixture,
called the homogenate, is centrifuged in a step-by-step fashion of increasing centrifugal force.
The denser material will form a pellet at lower centrifugal force (more...)
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4.1.3. Proteins Can Be Purified According to Solubility, Size, Charge, and Binding
Several thousand proteins have been purified in active form on the basis of such characteristics
as solubility, size, charge, and specific binding affinity. Usually, protein mixtures are subjected to
a series of separations, each based on a different property to yield a pure protein. At each step in
the purification, the preparation is assayed and the protein concentration is determined.
Substantial quantities of purified proteins, of the order of many milligrams, are needed to fully
elucidate their three-dimensional structures and their mechanisms of action. Thus, the overall
yield is an important feature of a purification scheme. A variety of purification techniques are
Salting Out

Most proteins are less soluble at high salt concentrations, an effect called salting out. The salt
concentration at which a protein precipitates differs from one protein to another. Hence, salting
out can be used to fractionate proteins. For example, 0.8 M ammonium sulfate precipitates
fibrinogen, a blood-clotting protein, whereas a concentration of 2.4 M is needed to precipitate
serum albumin. Salting out is also useful for concentrating dilute solutions of proteins, including
active fractions obtained from other purification steps. Dialysis can be used to remove the salt if

Proteins can be separated from small molecules by dialysis through a semipermeable membrane,
such as a cellulose membrane with pores (Figure 4.2). Molecules having dimensions
significantly greater than the pore diameter are retained inside the dialysis bag, whereas smaller

molecules and ions traverse the pores of such a membrane and emerge in the dialysate outside
the bag. This technique is useful for removing a salt or other small molecule, but it will not
distinguish between proteins effectively.

Figure 4.2

Dialysis. Protein molecules (red) are retained within the dialysis bag, whereas small molecules
(blue) diffuse into the surrounding medium.
Gel-Filtration Chromatography

More discriminating separations on the basis of size can be achieved by the technique of gelfiltration chromatography(Figure 4.3). The sample is applied to the top of a column consisting of
porous beads made of an insoluble but highly hydrated polymer such as dextran or agarose
(which are carbohydrates) or polyacrylamide. Sephadex, Sepharose, and Bio-gel are commonly
used commercial preparations of these beads, which are typically 100 m (0.1 mm) in diameter.
Small molecules can enter these beads, but large ones cannot. The result is that small molecules
are distributed in the aqueous solution both inside the beads and between them, whereas large
molecules are located only in the solution between the beads. Large molecules flow more rapidly
through this column and emerge first because a smaller volume is accessible to them. Molecules
that are of a size to occasionally enter a bead will flow from the column at an intermediate
position, and small molecules, which take a longer, tortuous path, will exit last.

Figure 4.3

Gel Filtration Chromatography. A mixture of proteins in a small volume is applied to a column

filled with porous beads. Because large proteins cannot enter the internal volume of the beads,
they emerge sooner than do small ones.

Ion-Exchange Chromatography

Proteins can be separated on the basis of their net charge by ion-exchange chromatography. If a
protein has a net positive charge at pH 7, it will usually bind to a column of beads containing
carboxylate groups, whereas a negatively charged protein will not (Figure 4.4). A positively
charged protein bound to such a column can then be eluted (released) by increasing the
concentration of sodium chloride or another salt in the eluting buffer because sodium ions
compete with positively charged groups on the protein for binding to the column. Proteins that
have a low density of net positive charge will tend to emerge first, followed by those having a
higher charge density. Positively charged proteins (cationic proteins) can be separated on
negatively charged carboxymethyl-cellulose (CM-cellulose) columns. Conversely, negatively
charged proteins (anionic proteins) can be separated by chromatography on positively charged
diethylaminoethyl-cellulose (DEAE-cellulose) columns.

Figure 4.4

Ion-Exchange Chromatography. This technique separates proteins mainly according to their net
Affinity Chromatography

Affinity chromatography is another powerful and generally applicable means of purifying

proteins. This technique takes advantage of the high affinity of many proteins for specific
chemical groups. For example, the plant protein concanavalin A can be purified by passing a
crude extract through a column of beads containing covalently attached glucose residues.

Concanavalin A binds to such a column because it has affinity for glucose, whereas most other
proteins do not. The bound concanavalin A can then be released from the column by adding a
concentrated solution of glucose. The glucose in solution displaces the column-attached glucose
residues from binding sites on concanavalin A (Figure 4.5). Affinity chromatography is a
powerful means of isolating transcription factors, proteins that regulate gene expression by
binding to specific DNA sequences. A protein mixture is percolated through a column containing
specific DNA sequences attached to a matrix; proteins with a high affinity for the sequence will
bind and be retained. In this instance, the transcription factor is released by washing with a
solution containing a high concentration of salt. In general, affinity chromatography can be
effectively used to isolate a protein that recognizes group X by (1) covalently attaching X or a
derivative of it to a column, (2) adding a mixture of proteins to this column, which is then
washed with buffer to remove unbound proteins, and (3) eluting the desired protein by adding a
high concentration of a soluble form of X or altering the conditions to decrease binding affinity.
Affinity chromatography is most effective when the interaction of the protein and the molecule
that is used as the bait is highly specific.

Figure 4.5

Affinity Chromatography. Affinity chromatography of concanavalin A (shown in yellow) on a

solid support containing covalently attached glucose residues (G).
High-Pressure Liquid Chromatography

The resolving power of all of the column techniques can be improved substantially through the
use of a technique calledhigh-pressure liquid chromatography (HPLC), which is an enhanced
version of the column techniques already discussed. The column materials themselves are much
more finely divided and, as a consequence, there are more interaction sites and thus greater
resolving power. Because the column is made of finer material, pressure must be applied to the

column to obtain adequate flow rates. The net result is high resolution as well as rapid separation
(Figure 4.6).

Figure 4.6

High-Pressure Liquid Chromatography (HPLC). Gel filtration by HPLC clearly defines the
individual proteins because of its greater resolving power: (1) thyroglobulin (669 kd), (2)
catalase (232 kd), (3) bovine serum albumin (67 kd), (4) ovalbumin (43 kd), (more...)
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4.1.4. Proteins Can Be Separated by Gel Electrophoresis and Displayed

How can we tell whether a purification scheme is effective? One way is to ascertain that the
specific activity rises with each purification step. Another is to visualize the effectiveness by
displaying the proteins present at each step. The technique of electrophoresis makes the latter
method possible.
Gel Electrophoresis

A molecule with a net charge will move in an electric field. This phenomenon,
termed electrophoresis, offers a powerful means of separating proteins and other
macromolecules, such as DNA and RNA. The velocity of migration (v) of a protein (or any
molecule) in an electric field depends on the electric field strength (E), the net charge on the
protein (z),and the frictional coefficient (f).

The electric force Ez driving the charged molecule toward the oppositely charged electrode is
opposed by the viscous drag fv arising from friction between the moving molecule and the

medium. The frictional coefficient f depends on both the mass and shape of the migrating
molecule and the viscosity () of the medium. For a sphere of radius r,

Electrophoretic separations are nearly always carried out in gels (or on solid supports such as
paper) because the gel serves as a molecular sieve that enhances separation (Figure 4.7).
Molecules that are small compared with the pores in the gel readily move through the gel,
whereas molecules much larger than the pores are almost immobile. Intermediate-size molecules
move through the gel with various degrees of facility. Electrophoresis is performed in a thin,
vertical slab of polyacrylamide. The direction of flow is from top to bottom. Polyacrylamide
gels, formed by the polymerization of acrylamide and cross-linked by methylenebisacrylamide,
are choice supporting media for electrophoresis because they are chemically inert and are readily
formed (Figure 4.8). Electrophoresis is the opposite of gel filtration in that all of the molecules,
regardless of size, are forced to move through the same matrix. The gel behaves as one bead of a
gel-filtration column.

Figure 4.7

Polyacrylamide Gel Electrophoresis. (A) Gel electrophoresis apparatus. Typically, several

samples undergo electrophoresis on one flat polyacrylamide gel. A microliter pipette is used to
place solutions of proteins in the wells of the slab. A cover is (more...)

Figure 4.8

Formation of a Polyacrylamide Gel. A three-dimensional mesh is formed by co-polymerizing

activated monomer (blue) and cross-linker (red).
Proteins can be separated largely on the basis of mass by electrophoresis in a polyacrylamide gel
under denaturing conditions. The mixture of proteins is first dissolved in a solution of sodium
dodecyl sulfate (SDS), an anionic detergent that disrupts nearly all noncovalent interactions in

native proteins. Mercaptoethanol (2-thioethanol) or dithiothreitol also is added to reduce

disulfide bonds. Anions of SDS bind to main chains at a ratio of about one SDS anion for every
two amino acid residues. This complex of SDS with a denatured protein has a large net negative
charge that is roughly proportional to the mass of the protein. The negative charge acquired on
binding SDS is usually much greater than the charge on the native protein; this native charge is
thus rendered insignificant. The SDS-protein complexes are then subjected to electrophoresis.
When the electrophoresis is complete, the proteins in the gel can be visualized by staining them
with silver or a dye such as Coomassie blue, which reveals a series of bands (Figure 4.9).
Radioactive labels can be detected by placing a sheet of x-ray film over the gel, a procedure
called autoradiography.

Figure 4.9

Staining of Proteins After Electrophoresis. Proteins subjected to electrophoresis on an SDSpolyacrylamide gel can be visualized by staining with Coomassie blue. [Courtesy of Kodak
Scientific Imaging Systems.]
Small proteins move rapidly through the gel, whereas large proteins stay at the top, near the
point of application of the mixture. The mobility of most polypeptide chains under these

conditions is linearly proportional to the logarithm of their mass (Figure 4.10). Some
carbohydrate-rich proteins and membrane proteins do not obey this empirical relation, however.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is rapid, sensitive, and capable of a high
degree of resolution. As little as 0.1 g (~2 pmol) of a protein gives a distinct band when stained
with Coomassie blue, and even less (~0.02 g) can be detected with a silver stain. Proteins that
differ in mass by about 2% (e.g., 40 and 41 kd, arising from a difference of about 10 residues)
can usually be distinguished.

Figure 4.10

Electrophoresis Can Determine Mass. The electrophoretic mobility of many proteins in SDSpolyacrylamide gels is inversely proportional to the logarithm of their mass. [After K. Weber and
M. Osborn, The Proteins, vol. 1, 3d ed. (Academic Press, 1975), (more...)
We can examine the efficacy of our purification scheme by analyzing a part of each fraction by
SDS-PAGE. The initial fractions will display dozens to hundreds of proteins. As the purification
progresses, the number of bands will diminish, and the prominence of one of the bands should
increase. This band will correspond to the protein of interest.
Isoelectric Focusing

Proteins can also be separated electrophoretically on the basis of their relative contents of acidic
and basic residues. Theisoelectric point (pl) of a protein is the pH at which its net charge is zero.
At this pH, its electrophoretic mobility is zero because z in equation 1 is equal to zero. For
example, the pI of cytochrome c, a highly basic electron-transport protein, is 10.6, whereas that
of serum albumin, an acidic protein in blood, is 4.8. Suppose that a mixture of proteins
undergoes electrophoresis in a pH gradient in a gel in the absence of SDS. Each protein will
move until it reaches a position in the gel at which the pH is equal to the pI of the protein. This
method of separating proteins according to their isoelectric point is called isoelectric focusing.
The pH gradient in the gel is formed first by subjecting a mixture of polyampholytes(small
multicharged polymers) having many pI values to electrophoresis. Isoelectric focusing can
readily resolve proteins that differ in pI by as little as 0.01, which means that proteins differing
by one net charge can be separated (Figure 4.11).

Figure 4.11

The Principle of Isoelectric Focusing. A pH gradient is established in a gel before loading the
sample. (A) The sample is loaded and voltage is applied. The proteins will migrate to their
isoelectric pH, the location at which they have no net charge. (more...)
Two-Dimensional Electrophoresis

Isoelectric focusing can be combined with SDS-PAGE to obtain very high resolution
separations. A single sample is first subjected to isoelectric focusing. This single-lane gel is then
placed horizontally on top of an SDS-polyacrylamide slab. The proteins are thus spread across
the top of the polyacrylamide gel according to how far they migrated during isoelectric focusing.
They then undergo electrophoresis again in a perpendicular direction (vertically) to yield a
twodimensional pattern of spots. In such a gel, proteins have been separated in the horizontal
direction on the basis of isoelectric point and in the vertical direction on the basis of mass. It is
remarkable that more than a thousand different proteins in the bacterium Escherichia coli can be
resolved in a single experiment by two-dimensional electrophoresis (Figure 4.12).

Figure 4.12

Two-Dimensional Gel Electrophoresis. (A) A protein sample is initially fractionated in one

dimension by isoelectric focusing as described in Figure 4.11. The isoelectric focusing gel is then
attached to an SDS-polyacrylamide gel, and electrophoresis is (more...)
Proteins isolated from cells under different physiological conditions can be subjected to twodimensional electrophoresis, followed by an examination of the intensity of the signals. In this
way, particular proteins can be seen to increase or decrease in concentration in response to the
physiological state. How can we tell what protein is being regulated? A former drawback to the
power of the two-dimensional gel is that, although many proteins are displayed, they are not
identified. It is now possible to identify proteins by coupling two-dimensional gel electrophoresis
with mass spectrometric techniques. We will consider these techniques when we examine how
the mass of a protein is determined (Section 4.1.7).

Go to:

4.1.5. A Protein Purification Scheme Can Be Quantitatively Evaluated

To determine the success of a protein purification scheme, we monitor the procedure at each step
by determining specific activity and by performing an SDS-PAGE analysis. Consider the results
for the purification of a fictitious protein, summarized in Table 4.1 and Figure 4.13. At each step,
the following parameters are measured:

Table 4.1

Quantification of a purification protocol for a fictitious protein.

Figure 4.13

Electrophoretic Analysis of a Protein Purification. The purification scheme in Table 4.1 was
analyzed by SDS-PAGE. Each lane contained 50 g of sample. The effectiveness of the
purification can be seen as the band for the protein of interest becomes (more...)

Total protein. The quantity of protein present in a fraction is obtained by

determining the protein concentration of a part of each fraction and
multiplying by the fraction's total volume.

Total activity. The enzyme activity for the fraction is obtained by measuring
the enzyme activity in the volume of fraction used in the assay and
multiplying by the fraction's total volume.

Specific activity. This parameter is obtained by dividing total activity by total


Yield. This parameter is a measure of the activity retained after each

purification step as a percentage of the activity in the crude extract. The
amount of activity in the initial extract is taken to be 100%.

Purification level. This parameter is a measure of the increase in purity and is

obtained by dividing the specific activity, calculated after each purification
step, by the specific activity of the initial extract.

As we see in Table 4.1, the first purification step, salt fractionation, leads to an increase in purity
of only 3-fold, but we recover nearly all the target protein in the original extract, given that the
yield is 92%. After dialysis to lower the high concentration of salt remaining from the salt
fractionation, the fraction is passed through an ion-exchange column. The purification now
increases to 9-fold compared with the original extract, whereas the yield falls to 77%. Molecular
exclusion chromatography brings the level of purification to 100-fold, but the yield is now at
50%. The final step is affinity chromatography with the use of a ligand specific for the target
enzyme. This step, the most powerful of these purification procedures, results in a purification
level of 3000-fold, while lowering the yield to 35%. The SDS-PAGE inFigure 4.13 shows that, if
we load a constant amount of protein onto each lane after each step, the number of bands
decreases in proportion to the level of purification, and the amount of protein of interest
increases as a proportion of the total protein present.
A good purification scheme takes into account both purification levels and yield. A high degree
of purification and a poor yield leave little protein with which to experiment. A high yield with
low purification leaves many contaminants (proteins other than the one of interest) in the fraction
and complicates the interpretation of experiments.
Go to:

4.1.6. Ultracentrifugation Is Valuable for Separating Biomolecules and Determining Their

We have already seen that centrifugation is a powerful and generally applicable method for
separating a crude mixture of cell components, but it is also useful for separating and analyzing
biomolecules themselves. With this technique, we can determine such parameters as mass and
density, learn something about the shape of a molecule, and investigate the interactions between
molecules. To deduce these properties from the centrifugation data, we need a mathematical
description of how a particle behaves in a centrifugal force.

A particle will move through a liquid medium when subjected to a centrifugal force. A
convenient means of quantifying the rate of movement is to calculate the sedimentation
coefficient, s, of a particle by using the following equation:

where m is the mass of the particle, is the partial specific volume (the reciprocal
of the particle density), is the density of the medium and f is the frictional
coefficient (a measure of the shape of the particle). The (1 - ) term is the buoyant
force exerted by liquid medium.

Sedimentation coefficients are usually expressed in Svedberg units (S), equal to 10-13 s. The
smaller the S value, the slower a molecule moves in a centrifugal field. The S values for a
number of biomolecules and cellular components are listed in Table 4.2 and Figure 4.14.

Table 4.2

S values and molecular weights of sample proteins.

Figure 4.14

Density and Sedimentation Coefficients of Cellular Components. [After L. J. Kleinsmith and V.

M. Kish, Principles of Cell and Molecular Biology, 2d ed. (Harper Collins, 1995), p. 138.]
Several important conclusions can be drawn from the preceding equation:

The sedimentation velocity of a particle depends in part on its mass. A more massive
particle sediments more rapidly than does a less massive particle of the same shape and

Shape, too, influences the sedimentation velocity because it affects the viscous drag. The
frictional coefficient f of a compact particle is smaller than that of an extended particle of
the same mass. Hence, elongated particles sediment more slowly than do spherical ones
of the same mass.

A dense particle moves more rapidly than does a less dense one because the opposing
buoyant force (1 - ) is smaller for the denser particle.

The sedimentation velocity also depends on the density of the solution. (). Particles sink
when < 1, float when > 1, and do not move when = 1.
A technique called zonal, band, or most commonly gradient centrifugation can be used to
separate proteins with different sedimentation coefficients. The first step is to form a density
gradient in a centrifuge tube. Differing proportions of a low-density solution (such as 5%
sucrose) and a high-density solution (such as 20% sucrose) are mixed to create a linear gradient
of sucrose concentration ranging from 20% at the bottom of the tube to 5% at the top (Figure
4.15). The role of the gradient is to prevent connective flow. A small volume of a solution
containing the mixture of proteins to be separated is placed on top of the density gradient. When
the rotor is spun, proteins move through the gradient and separate according to their
sedimentation coefficients. The time and speed of the centrifugation is determined empirically.
The separated bands, or zones, of protein can be harvested by making a hole in the bottom of the
tube and collecting drops. The drops can be measured for protein content and catalytic activity or
another functional property. This sedimentation-velocity technique readily separates proteins
differing in sedimentation coefficient by a factor of two or more.

Figure 4.15

Zonal Centrifugation. The steps are as follows: (A) form a density gradient, (B) layer the sample
on top of the gradient, (C) place the tube in a swinging-bucket rotor and centrifuge it, and (D)
collect the samples. [After D. Freifelder,Physical Biochemistry, (more...)
The mass of a protein can be directly determined by sedimentation equilibrium, in which a
sample is centrifuged at relatively low speed so that sedimentation is counterbalanced by
diffusion. The sedimentation-equilibrium technique for determining mass is very accurate and
can be applied under nondenaturing conditions in which the native quaternary structure of
multimeric proteins is preserved. In contrast, SDS-polyacrylamide gel electrophoresis (Section
4.1.4) provides an estimate of the mass of dissociated polypeptide chains
under denaturing conditions. Note that, if we know the mass of the dissociated components of a
multimeric protein as determined by SDS-polyacrylamide analysis and the mass of the intact
multimeric protein as determined by sedimentation equilibrium analysis, we can determine how
many copies of each polypeptide chain is present in the multimeric protein.

Figure 4.1Differential Centrifugation

Cells are disrupted in a homogenizer and the resulting mixture, called the homogenate, is
centrifuged in a step-by-step fashion of increasing centrifugal force. The denser material will
form a pellet at lower centrifugal force than will the less-dense material. The isolated fractions
can be used for further purification. [Photographs courtesy of S. Fleischer and B. Fleischer.]

Structural Biochemistry/Proteins/Purification
< Structural Biochemistry | Proteins
This page may need to be reviewed for quality.

General Information[edit]
Protein Purification is the process of separating proteins for individual analysis. Protein purification
is the second step of studying proteins, the first being the process of an assay. An assay is a
procedure to measure the activity enzyme activity thus confirming the presence of the protein or
proteins in interest. Popular assays include Western Blottingand ELISA(Enzyme-linked
immunosorbent assay). Before the purification process, Cell Disruption is utilized to homogenize the
cell's content. After the cell has been opened up, the process of purifying proteins from one another
and the other organelles can be approached in several different methods. Protein mixtures are
normally separated multiple times, each based on a different property, such as:



Molecular Weight


Binding affinity

The intended reason for purifying a specific protein governs the level and degree of protein
purification. At times, a sample of protein that is only moderately purified suffices for its intended
application; however, other situations require a higher degree of purification, especially if the

fundamental ambition is to study the characteristics and tendencies of the specific protein in interest.
By considering solubility, size, molecular weight, charge, and binding affinity, the goal of the scientist
that conducts protein purification is to find a level of purification necessary and create a protein yield
that is ample for further research and application. This means using the fewest amount of steps in
order to keep the yield high, as each protein purification step incurs a degree of product loss.
Therefore two factors serve as obstacles in protein purification: yield and purification level. The main
goal of each protein purification project falls under two categories: analytical (for studying and
research purposes) and preparative (for production and creation of commercial products).
There are many methods of purification including:

Differential Centrifugation

Salting Out

High Pressure Liquid Chromatography

Gel-Filtration Chromatography

Ion-Exchange Chromatography

Affinity Chromatography

Hydrophobic Interaction Chromatography

Fast Protein Liquid Chromatography

Isoelectric Focusing



Proteins Purification Methods

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Electrop ctric Dimensi
Interacti horesis Focus






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Protein purification
From Wikipedia, the free encyclopedia

Protein purification is a series of processes intended to isolate one or a few proteins from a
complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the
characterization of the function, structure and interactions of the protein of interest. The purification

process may separate the protein and non-protein parts of the mixture, and finally separate the
desired protein from all other proteins. Separation of one protein from all others is typically the most
laborious aspect of protein purification. Separation steps usually exploit differences in protein size,
physico-chemical properties, binding affinity and biological activity.
The methods used in protein purification can roughly be divided into analytical and preparative
methods. The distinction is not exact, but the deciding factor is the amount of protein that can
practically be purified with that method. Analytical methods aim to detect and identify a protein in a
mixture, whereas preparative methods aim to produce large quantities of the protein for other
purposes, such as structural biology or industrial use. In general, the preparative methods can be
used in analytical applications, but not the other way around.

1 Purpose

2 Preliminary Steps

2.1 Extraction

2.2 Precipitation and differential solubilization

2.3 Ultracentrifugation

3 Purification Strategies

3.1 Size exclusion chromatography

3.2 Separation based on charge or hydrophobicity

3.3 Affinity chromatography

3.3.1 Metal binding

3.3.2 Immunoaffinity chromatography

3.3.3 Purification of a tagged protein

3.4 HPLC
4 Concentration of the purified protein

4.1 Lyophilization

4.2 Ultrafiltration
5 Evaluating purification yield

6 Analytical

6.1 Denaturing-Condition Electrophoresis

6.2 Non-Denaturing-Condition Electrophoresis

7 See also

8 References

9 External links

Protein purification is either preparative or analytical. Preparative purifications aim to produce a
relatively large quantity of purified proteins for subsequent use. Examples include the preparation of
commercial products such as enzymes (e.g. lactase), nutritional proteins (e.g. soy protein isolate),
and certain biopharmaceuticals (e.g. insulin).Analytical purification produces a relatively small
amount of a protein for a variety of research or analytical purposes, including identification,
quantification, and studies of theprotein's structure, post-translational modifications and
function. Pepsin and urease were the first proteins purified to the point that they could be

Recombinant bacteria can be grown in a flask containing growth media.

Preliminary Steps[edit]

Depending on the source, the protein has to be brought into solution by breaking the tissue or cells
containing it. There are several methods to achieve this: Repeated freezing and
thawing, sonication, homogenization by high pressure, filtration, or permeabilization by organic
solvents. The method of choice depends on how fragile the protein is and how sturdy the cells are.
Usually for most of the conventional purposes, column chromatography is used to achieve
purification. After this extraction process soluble proteins will be in the solvent, and can be separated
from cell membranes, DNA etc. by centrifugation. The extraction process also extracts proteases,
which will start digesting the proteins in the solution. If the protein is sensitive to proteolysis, it is
usually desirable to proceed quickly, and keep the extract cooled, to slow down proteolysis. s

Precipitation and differential solubilization[edit]

Main article: Ammonium sulfate precipitation
In bulk protein purification, a common first step to isolate proteins is precipitation with ammonium
sulphate (NH4)2SO4. This is performed by adding increasing amounts of ammonium sulphate and
collecting the different fractions of precipitate protein. Ammonium sulphate can be removed by
dialysis.The hydrophobic groups on the proteins gets exposed to the atmosphere and it attracts
other protein hydrophobic groups and gets aggregated. Protein precipitated will be large enough to
be visible. One advantage of this method is that it can be performed inexpensively with very large
The first proteins to be purified are water-soluble proteins. Purification of integral membrane
proteins requires disruption of the cell membrane in order to isolate any one particular protein from
others that are in the same membrane compartment. Sometimes a particular membrane fraction can
be isolated first, such as isolating mitochondria from cells before purifying a protein located in a
mitochondrial membrane. A detergent such as sodium dodecyl sulfate (SDS) can be used to
dissolve cell membranes and keep membrane proteins in solution during purification; however,
because SDS causes denaturation, milder detergents such as Triton X-100 or CHAPS can be used
to retain the protein's native conformation during complete purification.

Main article: Ultracentrifuge
Centrifugation is a process that uses centrifugal force to separate mixtures of particles of varying
masses or densities suspended in a liquid. When a vessel (typically a tube or bottle) containing a
mixture of proteins or other particulate matter, such as bacterial cells, is rotated at high speeds,
the inertia of each particle yields an outward force proportional to its mass. The tendency of a given
particle to move through the liquid because of this force is offset by the resistance the liquid exerts
on the particle. The net effect of "spinning" the sample in a centrifuge is that massive, small, and
dense particles move outward faster than less massive particles or particles with more "drag" in the
liquid. When suspensions of particles are "spun" in a centrifuge, a "pellet" may form at the bottom of
the vessel that is enriched for the most massive particles with low drag in the liquid.

Non-compacted particles remain mostly in the liquid called "supernatant" and can be removed from
the vessel thereby separating the supernatant from the pellet. The rate of centrifugation is
determined by the angular acceleration applied to the sample, typically measured in comparison to
the g. If samples are centrifuged long enough, the particles in the vessel will reach equilibrium
wherein the particles accumulate specifically at a point in the vessel where their buoyant density is
balanced with centrifugal force. Such an "equilibrium" centrifugation can allow extensive purification
of a given particle.
Sucrose gradient centrifugation a linear concentration gradient of sugar (typically sucrose,
glycerol, or a silica based density gradient media, like Percoll) is generated in a tube such that the
highest concentration is on the bottom and lowest on top. Percoll is a trademark owned by GE
Healthcare companies. A protein sample is then layered on top of the gradient and spun at high
speeds in an ultracentrifuge. This causes heavy macromolecules to migrate towards the bottom of
the tube faster than lighter material. During centrifugation in the absence of sucrose, as particles
move farther and farther from the center of rotation, they experience more and more centrifugal force
(the further they move, the faster they move). The problem with this is that the useful separation
range of within the vessel is restricted to a small observable window. Spinning a sample twice as
long doesn't mean the particle of interest will go twice as far, in fact, it will go significantly further.
However, when the proteins are moving through a sucrose gradient, they encounter liquid of
increasing density and viscosity. A properly designed sucrose gradient will counteract the increasing
centrifugal force so the particles move in close proportion to the time they have been in the
centrifugal field. Samples separated by these gradients are referred to as "rate zonal"
centrifugations. After separating the protein/particles, the gradient is then fractionated and collected.

Purification Strategies[edit]

Chromatographic equipment. Here set up for a size exclusion chromatography. The buffer is pumped through
the column (right) by a computer controlled device.

Choice of a starting material is key to the design of a purification process. In a plant or animal, a
particular protein usually isn't distributed homogeneously throughout the body; different organs or
tissues have higher or lower concentrations of the protein. Use of only the tissues or organs with the
highest concentration decreases the volumes needed to produce a given amount of purified protein.
If the protein is present in low abundance, or if it has a high value, scientists may use recombinant
DNA technology to develop cells that will produce large quantities of the desired protein (this is
known as an expression system). Recombinant expression allows the protein to be tagged, e.g. by
a His-tag, to facilitate purification, which means that the purification can be done in fewer steps. In
addition, recombinant expression usually starts with a higher fraction of the desired protein than is
present in a natural source.
An analytical purification generally utilizes three properties to separate proteins. First, proteins may
be purified according to their isoelectric points by running them through a pH graded gel or an ion
exchange column. Second, proteins can be separated according to their size or molecular weight
via size exclusion chromatography or by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel
electrophoresis) analysis. Proteins are often purified by using 2D-PAGE and are then analysed
by peptide mass fingerprinting to establish the protein identity. This is very useful for scientific
purposes and the detection limits for protein are nowadays very low and nanogram amounts of
protein are sufficient for their analysis. Thirdly, proteins may be separated by polarity/hydrophobicity
via high performance liquid chromatography or reversed-phase chromatography.
Usually a protein purification protocol contains one or more chromatographic steps. The basic
procedure in chromatography is to flow the solution containing the protein through a column packed
with various materials. Different proteins interact differently with the column material, and can thus
be separated by the time required to pass the column, or the conditions required to elute the protein
from the column. Usually proteins are detected as they are coming off the column by their
absorbance at 280 nm. Many different chromatographic methods exist:

Size exclusion chromatography[edit]

Main article: Gel permeation chromatography
Chromatography can be used to separate protein in solution or denaturing conditions by using
porous gels. This technique is known as size exclusion chromatography. The principle is that smaller
molecules have to traverse a larger volume in a porous matrix. Consequentially, proteins of a certain
range in size will require a variable volume of eluent (solvent) before being collected at the other end
of the column of gel.
In the context of protein purification, the eluent is usually pooled in different test tubes. All test tubes
containing no measurable trace of the protein to purify are discarded. The remaining solution is thus
made of the protein to purify and any other similarly-sized proteins.

Separation based on charge or hydrophobicity[edit]

Hydrophobic Interaction Chromatography

HIC media is amphiphilic, with both hydrophobic and hydrophilic regions, allowing for separation of
proteins based on their surface hydrophobicity. In pure water, the interactions between the resin and
the hydrophobic regions of protein would be very weak, but this interaction is enhanced by applying
a protein sample to HIC resin in high ionic strength buffer. The ionic strength of the buffer is then
reduced to elute proteins in order of decreasing hydrophobicity.[2]
Ion exchange chromatography
Main article: Ion exchange chromatography
Ion exchange chromatography separates compounds according to the nature and degree of their
ionic charge. The column to be used is selected according to its type and strength of charge. Anion
exchange resins have a positive charge and are used to retain and separate negatively charged
compounds, while cation exchange resins have a negative charge and are used to separate
positively charged molecules.
Before the separation begins a buffer is pumped through the column to equilibrate the opposing
charged ions. Upon injection of the sample, solute molecules will exchange with the buffer ions as
each competes for the binding sites on the resin. The length of retention for each solute depends
upon the strength of its charge. The most weakly charged compounds will elute first, followed by
those with successively stronger charges. Because of the nature of the separating mechanism, pH,
buffer type, buffer concentration, and temperature all play important roles in controlling the
Ion exchange chromatography is a very powerful tool for use in protein purification and is frequently
used in both analytical and preparative separations.

Nickel-affinity column. The resin is blue since it has bound nickel.

Affinity chromatography[edit]
Main article: Affinity chromatography
Affinity Chromatography is a separation technique based upon molecular conformation, which
frequently utilizes application specific resins. These resins have ligands attached to their surfaces
which are specific for the compounds to be separated. Most frequently, these ligands function in a
fashion similar to that of antibody-antigen interactions. This "lock and key" fit between the ligand and
its target compound makes it highly specific, frequently generating a single peak, while all else in the
sample is unretained.
Many membrane proteins are glycoproteins and can be purified by lectin affinity chromatography.
Detergent-solubilized proteins can be allowed to bind to a chromatography resin that has been

modified to have a covalently attached lectin. Proteins that do not bind to the lectin are washed away
and then specifically bound glycoproteins can be eluted by adding a high concentration of a sugar
that competes with the bound glycoproteins at the lectin binding site. Some lectins have high affinity
binding to oligosaccharides of glycoproteins that is hard to compete with sugars, and bound
glycoproteins need to be released by denaturing the lectin.
Metal binding[edit]

Schematic showing the steps involved in a metal binding strategy for protein purification. The use of nickel
immobilized withNitrilotriacetic acid (NTA) is shown here.

Main article: Polyhistidine-tag

A common technique involves engineering a sequence of 6 to 8 histidines into the N- or Cterminal of the protein. The polyhistidine binds strongly to divalent metal ions such
as nickel and cobalt. The protein can be passed through a column containing immobilized nickel
ions, which binds the polyhistidine tag. All untagged proteins pass through the column. The protein
can be eluted with imidazole, which competes with the polyhistidine tag for binding to the column, or
by a decrease in pH (typically to 4.5), which decreases the affinity of the tag for the resin. While this
procedure is generally used for the purification of recombinant proteins with an engineered affinity
tag (such as a 6xHis tag or Clontech's HAT tag), it can also be used for natural proteins with an
inherent affinity for divalent cations.
Immunoaffinity chromatography[edit]

A HPLC. From left to right: A pumping device generating a gradient of two different solvents, a steel enforced
column and an apparatus for measuring the absorbance.

Main article: Immunoaffinity chromatography

Immunoaffinity chromatography uses the specific binding of an antibody to the target protein to
selectively purify the protein. The procedure involves immobilizing an antibody to a column material,
which then selectively binds the protein, while everything else flows through. The protein can be
eluted by changing the pH or the salinity. Because this method does not involve engineering in a tag,
it can be used for proteins from natural sources.[3]
Purification of a tagged protein[edit]
Another way to tag proteins is to engineer an antigen peptide tag onto the protein, and then purify
the protein on a column or by incubating with a loose resin that is coated with an
immobilized antibody. This particular procedure is known
as immunoprecipitation.Immunoprecipitation is quite capable of generating an extremely specific
interaction which usually results in binding only the desired protein. The purified tagged proteins can
then easily be separated from the other proteins in solution and later eluted back into clean solution.
When the tags are not needed anymore, they can be cleaved off by a protease. This often involves
engineering a protease cleavage site between the tag and the protein.

Main article: High performance liquid chromatography
High performance liquid chromatography or high pressure liquid chromatography is a form of
chromatography applying high pressure to drive the solutes through the column faster. This means
that the diffusion is limited and the resolution is improved. The most common form is "reversed
phase" HPLC, where the column material is hydrophobic. The proteins are eluted by a gradient of
increasing amounts of an organic solvent, such as acetonitrile. The proteins elute according to their
hydrophobicity. After purification by HPLC the protein is in a solution that only
contains volatile compounds, and can easily be lyophilized.[4] HPLC purification frequently results in
denaturation of the purified proteins and is thus not applicable to proteins that do not spontaneously

Concentration of the purified protein[edit]

A selectively permeable membrane can be mounted in a centrifuge tube. The buffer is forced through the
membrane by centrifugation, leaving the protein in the upper chamber.

At the end of a protein purification, the protein often has to be concentrated. Different methods exist.

If the solution doesn't contain any other soluble component than the protein in question the protein
can be lyophilized (dried). This is commonly done after an HPLC run. This simply removes all volatile
components, leaving the proteins behind.

Ultrafiltration concentrates a protein solution using selective permeable membranes. The function of
the membrane is to let the water and small molecules pass through while retaining the protein. The
solution is forced against the membrane by mechanical pump, gas pressure, or centrifugation.

Evaluating purification yield[edit]

The most general method to monitor the purification process is by running a SDS-PAGE of the
different steps. This method only gives a rough measure of the amounts of different proteins in the
mixture, and it is not able to distinguish between proteins with similar apparentmolecular weight.
If the protein has a distinguishing spectroscopic feature or an enzymatic activity, this property can be
used to detect and quantify the specific protein, and thus to select the fractions of the separation,
that contains the protein. If antibodies against the protein are available then western
blotting and ELISA can specifically detect and quantify the amount of desired protein. Some proteins
function as receptors and can be detected during purification steps by a ligand binding assay, often
using aradioactive ligand.
In order to evaluate the process of multistep purification, the amount of the specific protein has to be
compared to the amount of total protein. The latter can be determined by theBradford total protein

assay or by absorbance of light at 280 nm, however some reagents used during the purification
process may interfere with the quantification. For example,imidazole (commonly used for purification
of polyhistidine-tagged recombinant proteins) is an amino acid analogue and at low concentrations
will interfere with the bicinchoninic acid (BCA) assay for total protein quantification. Impurities in lowgrade imidazole will also absorb at 280 nm, resulting in an inaccurate reading of protein
concentration from UV absorbance.
Another method to be considered is Surface Plasmon Resonance (SPR). SPR can detect binding of
label free molecules on the surface of a chip. If the desired protein is an antibody, binding can be
translated directly to the activity of the protein. One can express the active concentration of the
protein as the percent of the total protein. SPR can be a powerful method for quickly determining
protein activity and overall yield. It is a powerful technology that requires an instrument to perform.

Denaturing-Condition Electrophoresis[edit]
Gel electrophoresis is a common laboratory technique that can be used both as preparative and
analytical method. The principle of electrophoresis relies on the movement of a charged ion in an
electric field. In practice, the proteins are denatured in a solution containing a detergent (SDS). In
these conditions, the proteins are unfolded and coated with negatively charged detergent molecules.
The proteins in SDS-PAGE are separated on the sole basis of their size.
In analytical methods, the protein migrate as bands based on size. Each band can be detected using
stains such as Coomassie blue dye or silver stain. Preparative methods to purify large amounts of
protein, require the extraction of the protein from the electrophoretic gel. This extraction may involve
excision of the gel containing a band, or eluting the band directly off the gel as it runs off the end of
the gel.
In the context of a purification strategy, denaturing condition electrophoresis provides an improved
resolution over size exclusion chromatography, but does not scale to large quantity of proteins in a
sample as well as the late chromatography columns.

Non-Denaturing-Condition Electrophoresis[edit]
An important non-denaturing electrophoretic procedure for isolating bioactive metalloproteins in
complex protein mixtures is QPNC-PAGE.

Protein analysis: Protein separation and characterization

Methods based on different solubility characteristics


Salting out

Isoelectric Precipitation

Solvent Fractionation

Denaturation of Contaminating Proteins

Separation based on different adsorption characteristics




Size Exclusion Chromatography

Separation by electrophoresis

Non-denaturing electrophoresis

Denaturing electrophoresis

In the previous section, techniques used to determine the total concentration of protein in a food were
discussed. Food analysts are also often interested in the type of proteins present in a food because each
protein has unique nutritional and physicochemical properties. Protein type is usually determined by
separating and isolating the individual proteins from a complex mixture of proteins, so that they can be
subsequently identified and characterized. Proteins are separated on the basis of differences in their
physicochemical properties, such as size, charge, adsorption characteristics, solubility and heat-stability.
The choice of an appropriate separation techniquedepends on a number of factors, including the
reasons for carrying out the analysis, the amount of sample available, the desired purity, the equipment
available, the type of proteins present and the cost. Large-scale methods are available for crude isolations
of large quantities of proteins, whereas small-scale methods are available for proteins that are expensive
or only available in small quantities.

One of the factors that must be considered during the separation procedure is the possibility that the
native three dimensional structure of the protein molecules may be altered. A prior knowledge of the
effects of environmental conditions on protein structure and interactions is extremely useful when
selecting the most appropriate separation technique. Firstly, because it helps determine the most suitable
conditions to use to isolate a particular protein from a mixture of proteins (e.g., pH, ionic strength,
solvent, temperature etc.), and secondly, because it may be important to choose conditions which will not
adversely affect the molecular structure of the proteins.
Methods based
on different

Proteins can be separated by exploiting differences in their solubility in aqueous solutions. The solubility
of a protein molecule is determined by its amino acid sequence because this determines its size, shape,
hydrophobicity and electrical charge. Proteins can be selectively precipitated or solubilized by altering the
pH, ionic strength, dielectric constant or temperature of a solution. These separation techniques are the
most simple to use when large quantities of sample are involved, because they are relatively quick,
inexpensive and are not particularly influenced by other food components. They are often used as the first
step in any separation procedure because the majority of the contaminating materials can be easily
Salting out

Proteins are precipitated from aqueous solutions when the salt concentration exceeds a critical level,
which is known as salting-out, because all the water is "bound" to the salts, and is therefore not available
to hydrate the proteins. Ammonium sulfate [(NH4)2SO4] is commonly used because it has a high watersolubility, although other neutral salts may also be used, e.g., NaCl or KCl. Generally a two-step
procedure is used to maximize the separation efficiency. In the first step, the salt is added at a
concentration just below that necessary to precipitate out the protein of interest. The solution is then
centrifuged to remove any proteins that are less soluble than the protein of interest. The salt concentration
is then increased to a point just above that required to cause precipitation of the protein. This precipitates
out the protein of interest (which can be separated by centrifugation), but leaves more soluble proteins in
solution. The main problem with this method is that large concentrations of salt contaminate the solution,
which must be removed before the protein can be resolubilzed, e.g., by dialysis or ultrafiltration.
Isoelectric Precipitation

The isoelectric point (pI) of a protein is the pH where the net charge on the protein is zero. Proteins tend
to aggregate and precipitate at their pI because there is no electrostatic repulsion keeping them apart.
Proteins have different isoelectric points because of their different amino acid sequences (i.e., relative
numbers of anionic and cationic groups), and thus they can be separated by adjusting the pH of a solution.
When the pH is adjusted to the pI of a particular protein it precipitates leaving the other proteins in

Solvent Fractionation

The solubility of a protein depends on the dielectric constant of the solution that surrounds it because this
alters the magnitude of the electrostatic interactions between charged groups. As the dielectric constant of
a solution decreases the magnitude of the electrostatic interactions between charged species increases.
This tends to decrease the solubility of proteins in solution because they are less ionized, and therefore the
electrostatic repulsion between them is not sufficient to prevent them from aggregating. The dielectric
constant of aqueous solutions can be lowered by adding water-soluble organic solvents, such as ethanol or
acetone. The amount of organic solvent required to cause precipitation depends on the protein and
therefore proteins can be separated on this basis. The optimum quantity of organic solvent required to
precipitate a protein varies from about 5 to 60%. Solvent fractionation is usually performed at 0 oC or
below to prevent protein denaturation caused by temperature increases that occur when organic solvents
are mixed with water.
Denaturation of Contaminating Proteins

Many proteins are denatured and precipitate from solution when heated above a certain temperature or by
adjusting a solution to highly acid or basic pHs. Proteins that are stable at high temperature or at extremes
of pH are most easily separated by this technique because contaminating proteins can be precipitated
while the protein of interest remains in solution.
Separation based on
different adsorption

Adsorption chromatography involves the separation of compounds by selective adsorption-desorption at a

solid matrix that is contained within a column through which the mixture passes. Separation is based on
the different affinities of different proteins for the solid matrix. Affinity and ion-exchange
chromatography are the two major types of adsorption chromatography commonly used for the separation
of proteins. Separation can be carried out using either an open column or high-pressure liquid
Ion Exchange Chromatography

Ion exchange chromatography relies on the reversible adsorption-desorption of ions in solution to a

charged solid matrix or polymer network. This technique is the most commonly used chromatographic
technique for protein separation. A positively charged matrix is called an anion-exchanger because it
binds negatively charged ions (anions). A negatively charged matrix is called a cation-exchanger because
it binds positively charged ions (cations). The buffer conditions (pH and ionic strength) are adjusted to
favor maximum binding of the protein of interest to the ion-exchange column. Contaminating proteins
bind less strongly and therefore pass more rapidly through the column. The protein of interest is then
eluted using another buffer solution which favors its desorption from the column (e.g., different pH or
ionic strength).
Affinity Chromatography

Affinity chromatography uses a stationary phase that consists of a ligand covalently bound to a solid
support. The ligand is a molecule that has a highly specific and unique reversible affinity for a particular
protein. The sample to be analyzed is passed through the column and the protein of interest binds to the

ligand, whereas the contaminating proteins pass directly through. The protein of interest is then eluted
using a buffer solution which favors its desorption from the column. This technique is the most efficient
means of separating an individual protein from a mixture of proteins, but it is the most expensive, because
of the need to have columns with specific ligands bound to them.
Both ion-exchange and affinity chromatography are commonly used to separate proteins and amino-acids
in the laboratory. They are used less commonly for commercial separations because they are not suitable
for rapidly separating large volumes and are relatively expensive.
Separation based
on size

Proteins can also be separated according to their size. Typically, the molecular weights of proteins vary
from about 10,000 to 1,000,000 daltons. In practice, separation depends on the Stokes radius of a protein,
rather than directly on its molecular weight. The Stokes radius is the average radius that a protein has in
solution, and depends on its three dimensional molecular structure. For proteins with the same molecular
weight the Stokes radius increases in the following order: compact globular protein < flexible randomcoil < rod-like protein.

Dialysis is used to separate molecules in solution by use of semipermeable membranes that permit the
passage of molecules smaller than a certain size through, but prevent the passing of larger molecules. A
protein solution is placed in dialysis tubing which is sealed and placed into a large volume of water or
buffer which is slowly stirred. Low molecular weight solutes flow through the bag, but the large
molecular weight protein molecules remain in the bag. Dialysis is a relatively slow method, taking up to
12 hours to be completed. It is therefore most frequently used in the laboratory. Dialysis is often used to
remove salt from protein solutions after they have been separated by salting-out, and to change buffers.

A solution of protein is placed in a cell containing a semipermeable membrane, and pressure is applied.
Smaller molecules pass through the membrane, whereas the larger molecules remain in the solution. The
separation principle of this technique is therefore similar to dialysis, but because pressure is applied
separation is much quicker. Semipermeable membranes with cutoff points between about 500 to 300,000
are available. That portion of the solution which is retained by the cell (large molecules) is called the
retentate, whilst that part which passes through the membrane (small molecules) forms part of the
ultrafiltrate. Ultrafiltration can be used to concentrate a protein solution, remove salts, exchange buffers
or fractionate proteins on the basis of their size. Ultrafiltration units are used in the laboratory and on a
commercial scale.
Size Exclusion Chromatography

This technique, sometimes known as gel filtration, also separates proteins according to their size. A
protein solution is poured into a column which is packed with porous beads made of a cross-linked
polymeric material (such as dextran or agarose). Molecules larger than the pores in the beads are
excluded, and move quickly through the column, whereas the movement of molecules which enter the

pores is retarded. Thus molecules are eluted off the column in order of decreasing size. Beads of different
average pore size are available for separating proteins of different molecular weights. Manufacturers of
these beads provide information about the molecular weight range that they are most suitable for
separating. Molecular weights of unknown proteins can be determined by comparing their elution
volumes Vo, with those determined using proteins of known molecular weight: a plot of elution volume
versus log(molecular weight) should give a straight line. One problem with this method is that the
molecular weight is not directly related to the Stokes radius for different shaped proteins.
Separation by

Electrophoresis relies on
differences in the migration of
charged molecules in a solution
when an electrical field is applied
across it. It can be used to
separate proteins on the basis of
their size, shape or charge.
An electrode apparatus consists of
a high-voltage supply, electrodes,
buffer, and a support for the buffer
such as filter paper, cellulose
acetate strips, polyacrylamide gel,
or a capillary tube. Open capillary
tubes are used for many types of
samples and the other supports
are usually used for biological
samples such as protein mixtures
or DNA fragments. After a
separation is completed the
support is stained to visualize the
separated components.

Electorphoresis support with

separated staines

Principle of electrophoresis

Non-denaturing Electrophoresis

In non-denaturing electrophoresis, a buffered solution of native proteins is poured onto a porous gel
(usually polyacrylamide, starch or agarose) and a voltage is applied across the gel. The proteins move
through the gel in a direction that depends on the sign of their charge, and at a rate that depends on the
magnitude of the charge, and the friction to their movement:
applied voltage x molecular charge
mobility =
molecular friction
Proteins may be positively or negatively charged in solution depending on their isoelectic points (pI) and
the pH of the solution. A protein is negatively charged if the pH is above the pI, and positively charged if
the pH is below the pI. The magnitude of the charge and applied voltage will determine how far proteins
migrate in a certain time. The higher the voltage or the greater the charge on the protein the further it will
move. The friction of a molecule is a measure of its resistance to movement through the gel and is largely
determined by the relationship between the effective size of the molecule, and the size of the pores in the
gel. The smaller the size of the molecule, or the larger the size of the pores in the gel, the lower the
resistance and therefore the faster a molecule moves through the gel. Gels with different porosity's can be
purchased from chemical suppliers, or made up in the laboratory. Smaller pores sizes are obtained by
using a higher concentration of cross-linking reagent to form the gel. Gels may be contained between two
parallel plates, or in cylindrical tubes. In non-denaturing electrophoresis the native proteins are separated
based on a combination of their charge, size and shape.
Denaturing Electrophoresis

In denaturing electrophoresis proteins are separated primarily on their molecular weight. Proteins are
denatured prior to analysis by mixing them with mercaptoethanol, which breaks down disulfide bonds,
and sodium dodecyl sulfate (SDS), which is an anionic surfactant that hydrophobically binds to protein
molecules and causes them to unfold because of the repulsion between negatively charged surfactant
head-groups. Each protein molecule binds approximately the same amount of SDS per unit length. Hence,
the charge per unit length and the molecular conformation is approximately similar for all proteins. As
proteins travel through a gel network they are primarily separated on the basis of their molecular weight
because their movement depends on the size of the protein molecule relative to the size of the pores in the
gel: smaller proteins moving more rapidly through the matrix than larger molecules. This type of
electrophoresis is commonly called sodium dodecyl sulfate -polyacrylamide gel electrophoresis, or SDSPAGE. To determine how far proteins have moved a tracking dye is added to the protein solution, e.g.,
bromophenol blue. This dye is a small charged molecule that migrates ahead of the proteins. After the
electrophoresis is completed the proteins are made visible by treating the gel with a protein dye such as
Coomassie Brilliant Blue or silver stain. The relative mobility of each protein band is calculated:
distance protein moves
Rm =

distance dye moves

Electrophoresis is often used to determine the protein composition of food products. The protein is
extracted from the food into solution, which is then separated using electrophoresis. SDS-PAGE is used to
determine the molecular weight of a protein by measuring Rm, and then comparing it with a calibration
curve produced using proteins of known molecular weight: a plot of log (molecular weight) against
relative mobility is usually linear. Denaturing electrophoresis is more useful for determining molecular
weights than non-denaturing electrophoresis, because the friction to movement does not depend on the
shape or original charge of the protein molecules.

From Wikipedia, the free encyclopedia


IUPAC name
Other names

CAS Registry Number





4470639 aldehydo form D-()Ribose



EC number


InChI=1/C5H10O5/c6-1-3(8)5(10)4(9)2-7/h1,3-5,710H,2H2/t3-,4+,5-/m0/s1 aldehydo form D-()-Ribose
Jmol-3D images

aldehydo form D-()-Ribose


5311110 aldehydo form D-()-Ribose

C([C@H]([C@H]([C@H](C=O)O)O)O)O aldehydo form D()-Ribose
Chemical formula


Molar mass

150.13 g/mol


white solid

Melting point

95 C (203 F; 368 K)

Solubility in water

very soluble

Chiral rotation([]D)

21.5 (H2O)
Related compounds



Related compounds


Except where otherwise noted, data are given for materials in

their standard state (at 25 C [77 F], 100 kPa).
verify (what is:

/ ?)

Infobox references

Ribose is an organic compound with the formula C5H10O5; specifically,

a pentose monosaccharide (simple sugar) with linear form H(C=O)(CHOH)4H, which has all
the hydroxyl groups on the same side in the Fischer projection.
The term may refer to either of two enantiomers. The term usually indicates D-ribose, which occurs
widely in nature and is discussed here. Its synthetic mirror image, L-ribose, is not found in nature.

was first reported in 1891 by Emil Fischer. It is a C'-2 carbon epimer of the sugar Darabinose (both isomers of which are named for their source, gum arabic) and ribose itself is named
as a transposition of the name of arabinose.[3]
The ribose -D-ribofuranose forms part of the backbone of RNA. It is related to deoxyribose, which is
found in DNA. Phosphorylatedderivatives of ribose such as ATP and NADH play central roles
in metabolism. cAMP and cGMP, formed from ATP and GTP, serve as secondary messengers in
some signalling pathways.

Ribose is an aldopentose (a monosaccharide containing five carbon atoms) that, in its open
chain form, has an aldehyde functional group at one end. In the conventional numbering scheme for
monosaccharides, the carbon atoms are numbered from C1' (in the aldehyde group) to C5'.
The deoxyribose derivative found in DNA differs from ribose by having a hydrogen atom in place of
thehydroxyl group at C2'. This hydroxyl group performs a function in RNA splicing.
Like many monosaccharides, ribose exists in an equilibrium among 5 formsthe linear form H
(C=O)(CHOH)4H and either of the two ring forms: alpha- or beta-ribofuranose ("C3'-endo"), with a
five-membered ring, and alpha- or beta-ribopyranose ("C2'-endo"), with a six-membered ring. The
beta-ribopyranose form predominates in aqueous solution.[4]
The "D-" in the name D-ribose refers to the stereochemistry of the chiral carbon atom farthest away
from the aldehyde group (C4'). InD-ribose, as in all D-sugars, this carbon atom has the same
configuration as in D-glyceraldehyde.





Relative abundance of different forms of ribose in solution: -D-ribopyranose (59%), -Dribopyranose (20%), -D-ribofuranose (13%), -D-ribofuranose (7%) and open chain (0.1%). [5]

In biology, D-ribose must be phosphorylated by the cell before it can be used. Ribokinase catalyzes
this reaction by converting D-ribose to D-ribose 5-phosphate. Once converted, D-ribose-5-phosphate
is available for the manufacturing of the amino acidstryptophan and histidine, or for use in
the pentose phosphate pathway. The absorption of D-ribose is 88100% in the small intestines (up to
200 mg/kg/h).[6]

MeselsonStahl experiment
From Wikipedia, the free encyclopedia

The MeselsonStahl experiment was an experiment by Matthew Meselson and Franklin Stahl in
1958 which supported the hypothesis that DNA replication wassemiconservative. In
semiconservative replication, when the double stranded DNA helix is replicated each of the two new
double-stranded DNA helixes consisted of one strand from the original helix and one newly

synthesized. It has been called "the most beautiful experiment in biology.[1]" Meselson and Stahl
decided the best way to tag the parent DNA would be to change one of the atoms in the parent DNA
molecule. Since nitrogen is found in the nitrogenous bases of each nucleotide, they decided to use
an isotope of nitrogen to distinguish between parent and newly-copied DNA. The isotope of nitrogen
had an extra neutron in the nucleus, which made it heavier.

1 Hypothesis

2 Experimental procedure and results

3 References

4 External links


A summary of the three postulated methods of DNA synthesis

Three hypotheses had been previously proposed for the method of replication of DNA.
In the semiconservative hypothesis, proposed by Watson and Crick, the two strands of a DNA
molecule separate during replication. Each strand then acts as a template for synthesis of a new
The conservative hypothesis proposed that the entire DNA molecule acted as a template for the
synthesis of an entirely new one. According to this model, histone proteins bind to the DNA,
revolving the strand and exposing the nucleotide bases (which normally line the interior) for
hydrogen bonding.[3]
The dispersive hypothesis is exemplified by a model proposed by Max Delbrck, which attempts to
solve the problem of unwinding the two strands of the double helix by a mechanism that breaks the
DNA backbone every 10 nucleotides or so, untwists the molecule, and attaches the old strand to the
end of the newly synthesized one. This would synthesize the DNA in short pieces alternating from
one strand to the other.[4]
Each of these three models makes a different prediction about the distribution of the "old" DNA in
molecules formed after replication. In the conservative hypothesis, after replication, one molecule is
the entirely conserved "old" molecule, and the other is all newly synthesized DNA. The

semiconservative hypothesis predicts that each molecule after replication will contain one old and
one new strand. The dispersive model predicts that each strand of each new molecule will contain a
mixture of old and new DNA.[5]

Experimental procedure and results[edit]

Nitrogen is a major constituent of DNA. 14N is by far the most abundant isotope of nitrogen, but DNA
with the heavier (but non-radioactive) 15N isotope is also functional.
E. coli were grown for several generations in a medium with 15N. When DNA is extracted from these
cells and centrifuged on a salt density gradient, the DNA separates out at the point at which its
density equals that of the salt solution. The DNA of the cells grown in 15N medium had a higher
density than cells grown in normal 14N medium. After that, E. coli cells with only 15N in their DNA were
transferred to a 14N medium and were allowed to divide; the progress of cell division was monitored
by microscopic cell counts and by colony assay.
DNA was extracted periodically and was compared to pure 14N DNA and 15N DNA. After one
replication, the DNA was found to have intermediate density. Since conservative replication would
result in equal amounts of DNA of the higher and lower densities (but no DNA of an intermediate
density), conservative replication was excluded. However, this result was consistent with both
semiconservative and dispersive replication. Semiconservative replication would result in doublestranded DNA with one strand of 15N DNA, and one of 14N DNA, while dispersive replication would
result in double-stranded DNA with both strands having mixtures of 15N and 14N DNA, either of which
would have appeared as DNA of an intermediate density.
The authors continued to sample cells as replication continued. DNA from cells after two replications
had been completed was found to consist of equal amounts of DNA with two different densities, one
corresponding to the intermediate density of DNA of cells grown for only one division in 14N medium,
the other corresponding to DNA from cells grown exclusively in 14N medium. This was inconsistent
with dispersive replication, which would have resulted in a single density, lower than the intermediate
density of the one-generation cells, but still higher than cells grown only in 14N DNA medium, as the
original 15N DNA would have been split evenly among all DNA strands. The result was consistent with
the semiconservative replication hypothesis.[6]

Department Of Biochemistry And Microbiology Biology Essay Essays Biology

... assumption that 1 mol of ribose will account for 1 mol of ATP within a sample
ofpure ATP. Ribose in an acidic solution will result in the formation of furfural which in turn
reacts with orcinol, generating a green colour. ... In this case differentialcentrifugation is the
focus. .... The sucrose isolation media was used as a blank.
4- Separation based on conformation
Plasmids are supercoiled molecules formed by partial unwinding of double helix of the
plasmid DNA during the plasmid replication process by enzymes called topoisomerases.
The supercoiled conformation can be maintained when both polynucleotide strands are
intact, hence called covalently closed-circular (ccc) DNA. If one of the polynucleotide
strands is broken, the double helix reverts to its normal relaxed state taking an
alternative conformation, called open-circular (oc). Super coiling is important in plasmid
preparation due to the easy separation of supercoiled molecules from non-supercoiled
The commonly used methods of separation based on conformation are as follows4- Alkaline denaturation method
This method is based on maintaining a very narrow pH range for the denaturation of
non-supercoiled DNA but not the supercoiled plasmid (Figure 4-
Addition of sodium hydroxideto cell extract or cleared lysate (pH12.0-12.5) results in
disruption of the hydrogen bonds of non-supercoiled DNA molecules.
As a result, the double helix unwinds and two polynucleotide chains separate.
Further addition of acid causes the aggregation of these denatured bacterial DNA
strands into a tangled mass which can be pelleted by centrifugation, leaving plasmid
DNA in the supernatant.
Most of the RNA and protein under defined conditions (specifically cell lysis by SDS
and neutralization with sodium acetate) can be removed by the centrifugation.

No requirement of organic extraction.

Figure 4- Separation of plasmid DNA by Alkaline denaturation method







Density gradient centrifugation can separate DNA, RNA and protein. It is a very
efficient method for obtaining pure plasmid DNA.

A density gradient is produced by centrifuging a solution of cesium chloride at a very

high speed which pulls the CsCl ions towards the bottom. This process is referred as
isopycnic centrifugation.
The DNA migrates to the point at which it has density similar to that of CsCI i.e.1.7
g/cm3 in the gradient.
In contrast, protein molecules having lower buoyant densities float at the top of the
tube whereas RNA gets pelleted at the bottom.
Density gradient centrifugation in the presence of ethidium bromide (EtBr) can be used
to separate supercoiled DNA from non-super coiled molecules. Ethidium bromide is an
intercalating dye that binds to DNA molecules causing partial unwinding of the double
helix. Supercoiled DNA have very little freedom to unwind due to absence of free ends
and bind to a limited amount of EtBr resulting in very less decrease in buoyant density
(0.085 g/cm3 ) than that of linear DNA (0.125 g/cm 3 ). As a result, they form a distinctb
and separated from the linear bacterial DNA. The EtBr bound to DNA is then extracted
by n-butanol and the CsCl is removed by dialysis.
4-1.4. Isolation and Purification of RNA
RNA (Ribonucleic acid) is a polymeric substance consisting of a long single-stranded
chain of phosphate and ribose units with the nitrogen bases adenine, guanine, cytosine
and uracil bonded to the ribose sugar present in living cells and many viruses. The steps
for preparation of RNA involve homogenization, phase separation, RNA precipitation,
washing and re-dissolving RNA.
The method for isolation and purification of RNA are as follows1. Organic extraction method
2. Filter-based, spin basket formats
3. Magnetic particle methods
4. Direct lysis method.
4-1.4.1. Organic extraction method
This method involves phase separation by addition and centrifugation of a mixture of a
solution containing phenol, chloroform and a chaotropic agent (guanidinium
thiocyanate) and aqueous sample. Guanidium thiocyanate results in the denaturation of
proteins and RNases, separating rRNA from ribosomes. Addition of chloroform forms a
colorless upper aqueous phase containing RNA, an interphase containing DNA and a
lower phenol-chloroform phase containing protein. RNA is collected from the upper
aqueous phase by alcohol (2-propanol or ethanol) precipitation followed by rehydration.

One of the advantages of this method is the stabilization of RNA and rapid denaturation
of nucleases. Besides advantages, it has several drawbacks such as it is difficult to
automate, needs labor and manual intensive processing, and use of chlorinated organic
4-1.4.2. Direct lysis methods
This method involves use of lysis buffer under specified conditions for the disruption of
sample and stabilization of nucleic acids. If desired, samples can also be purified from
stabilized lysates. This method eliminates the need of binding and elution from solid
surfaces and thus avoids bias and recovery efficiency effects.
Extremely fast and easy.
Highest ability for precise RNA representation.
Easy to work on very small samples.
Amenable to simple automation.
Unable to perform traditional analytical methods (e.g. spectrophotometric method).
Dilution-based (most useful with concentrated samples).
Potential for suboptimal performance unless developed/optimized with downstream
Potential for residual RNase activity if lysates are not handled properly.

Department Of Biochemistry And

Microbiology Biology Essay
Adenosine triphosphate is a mononucleotide with an adenine base and a ribose sugar to which three
phosphate groups are linked. The covalent bond between the second and the third phosphate group

is unstable and easily broken by hydrolysis, thereby releasing energy (Kent, 2000). ATP can
therefore store energy so as to be used to drive endergonic reactions through a process of
phosphorylation. Phosphorylation refers to the transfer of energy with the addition of a phosphate
group from ATP to an endergonic reaction allowing for the reaction to be driven forward. ATP is
therefore essential for life as it provides the much needed energy to drive a living system (Alters,
There are various ways to quantify ATP, one of which is through absorption spectroscopy by
ultraviolet light. The absorbance of light, at a given wavelength, by a substance with chromophore
properties results in an exponential drop in light intensity (Wilson and Walker, 2010). With the
knowledge of the substances' molar extinction coefficient or the measure of how strongly the
substances absorbs light at a given wavelength, the concentration of the substance can be
determined with the use of the Beer-Lamberts Law:, where A refers to absorbance at the given
wavelength, refers to the molar extinction coefficient, c the concentration of the sample and l the
distance light travelled through the sample (Bansal, 2003). Purine bases such as adenine, a key
component of ATP, absorb UV light between 260 and 275nm allowing for their quantification by this

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An additional method of quantifying ATP can be carried out indirectly by an assay procedure for
pentose sugar, namely ribose. As discussed earlier ATP contains one ribose molecule allowing for
the assumption that 1 mol of ribose will account for 1 mol of ATP within a sample of pure ATP. Ribose
in an acidic solution will result in the formation of furfural which in turn reacts with orcinol, generating
a green colour. The intensity of the green colour can therefore be used to spectrophotometrically
quantify ribose at 660nm and indirectly ATP, considering no additional pentose sugar contaminates
are present (Nigam, 2007).

Many applications require the quantification of protein, often in a high throughput manner as can be
expected within various laboratories. One such method involves the use of the bicinchoninic acid
(BCA) protein assay developed by Smith et al. The assay depends on the conversion of to under
alkaline conditions which in turn reacts with BCA resulting in an intense purple colour, which can be
quantified through spectroscopy techniques at an absorbance of 562nm. The production of in the
assay is a function of the protein concentration and the incubation time, allowing for the protein
quantification (Walker, 1994). In addition, fluorimetric protein quantification can be carried out with
the aid of a reactive compound; fluorescamine. Fluorescamine is a very sensitive fluorogenic
reagent which reacts with primary amides to form fluorescent pyrrolinones. This results in a greenyellow fluorescence which can be quantified as a function of protein amide groups (Rost, 1995),
such as the terminal protein amide groups and -amino group of lysine (Rosenthal and Wright,
Other general techniques often used within a biochemical laboratory are centrifugation techniques.
In this case differential centrifugation is the focus. The process is based upon the differing
sedimentation rates of biological particles as a function of differing size and density. For example,
this allows for the division of crude homogenates into various fractions of organelles, membrane
vesicles and other structural fragments by means of a stepwise increase in the applied centrifugal
field and centrifugation times (Wilson and Walker, 2010). One of the classic examples is the
stepwise separation of liver homogenate to isolate liver mitochondria, whereby the effectiveness of
the technique can be analyzed through a succinate dehydrogenase (SDH) assay. Succinate
dehydrogenase exists ubiquitously within mitochondria in all aerobic tissue whereby the enzyme
catalyses the breakdown of succinate to fumarate forming FADH in the process. The breakdown of
succinate can be used as a mitochondrial indicator by addition of blue/purple redox dye namely
dichlorophenol indophenols (DCPIP). Blue DCPIP acts as a hydrogen acceptor from FADH (i.e.
product of the SDH enzyme) resulting in a loss of colour during the process, which can be directly
quantified spectrophotometrically at an absorbance of 600nm and thereby related to mitochondrial
concentration (Nigam, 2007).
The aim of the experiment is to analyze various techniques to determine ATP and protein
concentrations of unknown ATP samples A. In addition, centrifugation techniques with a chicken liver
homogenate as a model will be addressed, creating fractions which are to be assayed for
mitochondrial activity.

Section A
Chemical Techniques

1. Quantification of ATP using Absorption by Ultraviolet Light

The quantification ATP of an unknown ATP sample A (group 1) was carried out by means of
ultraviolet absorption spectroscopy. The unknown sample of 0.45g was diluted to make a stock
solution of 10mg/ml and for further diluted as required for subsequent absorbance spectroscopy
procedures (e.g. 1:10 or 1:100 dilution). The absorbance spectrum from 220 to 310nm of the
unknown ATP sample was performed and the content of the ATP was determined from the optical
density at 295nm. The molar extinction coefficient of ATP at pH 7 at 295nm was taken to be.
2. Quantification of ATP using the Ribose Content
2ml of the unknown ATP sample A solution was mixed with 2ml of 1% orcinol which was dissolved in
0.1% FeCl in concentrated HCl. The mixture was prepared in test tubes and sealed with cotton
bungs. The mixtures were then heated for 30min in a boiling water bath. The mixture was then
cooled and then diluted to 4ml. The optical densities of the samples were then observed at 660nm. A
standard curve was also created ranging from 0.01 - 0.1 mg/ml whereby the unknown sample was
substituted for known concentrations of ribose. The whole procedure for determining the ribose
content was carried out in triplicate. Refer to Table 3.1 for assay procedure within the appendix.

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Biochemical Techniques - Protein

Determination Methods
1. VIS Microtiter Plate Quantification of Protein using the Bicichonic Acid (BCA) assay
A BCA assay procedure was carried out to determine the protein content of unknown ATP sample A
with the use of a 96-well VIS microtiter plate reader. A five point standard curve of known BSA was
created (0 -2 mg/ml). Refer below to Table 1.1 for BCA assay procedure. Refer to Table 3.2 of
appendix for the assay procedure.

Table 1.1: BCA assay for the determination of unknown ATP sample A within Bovine Serum Albumin
(BSA) standard curve
Volume (l)
Reagent A and Reagent B* (50:1)
Incubated at 37&deg;C for 30min. Absorbance read at A540nm
*Refer to appendix for constituents of reagent A and B
2. Fluorimetric Quantitation of Protein using the Reactive Compound Fluorescamine
A five point standard curve of BSA was constructed ranging from 0 - 500g/ml, diluted in phosphate
buffered saline (PBS) at pH 7.4 to determine the unknown ATP sample A protein concentration.
150l aliquots were then transferred to a black 96-well microtiter plate followed with the addition of
50l of 3mg/ml fluorescamine dissolved in acetone. The plate was then shaken for 1min followed by
fluorescence determination with a 355nm excitation filter and a 460nm emission filter. All samples
were carried out in triplicate. Refer to Table 3.3 of appendix for assay procedure.

Section B
Mitochondrial enzymatic activity was determined whereby 50g of chicken liver tissue was obtained
and minced before being diluted in 10ml of sucrose isolation medium (300mmol/l sucrose: 0.5mmol/
EDTA at pH 7.4) per gram of tissue. The sample was then homogenized with a co-axial
homogenizer. The mortar and pestle was then fixed to a drill resulting in relative mortar movement to
the pestle, rotating at 2400rpm for 8-10 complete strokes. Procedures were performed at 4&deg;C.
Centrifugation of the suspension was then carried out as observed in Figure 1.1. However, the last
centrifugation step was altered to be 40000xg for 120min as opposed to 100000xg for 120min due to
the rotor of ultracentrifuge only being rated to 40000xg.
*Note aliquots of each fraction (H1, S1, S2, S3, P1, P2, and P3) were retained

Fig. 1.1: Differential centrifugation procedure carried out on chicken liver homogenate
A succinate dehydrogenase (SDH) assay was performed on the homogenate, supernatant and pellet
samples obtained during differential centrifugation of the liver sample. Assay medium was created as
can be seen in the appendix (Table 3.4) to be used in the SDH assay. For each sample 2.8ml of the
assay medium was pre-warmed to 37&deg;C followed by the addition of 0.2ml of the dilute enzyme
within the homogenate, supernatant or pellet samples and the absorbance was determined at
600nm. This was taken to be time zero (t). The subsequent absorbance values were recorded at
1min time intervals for a total of 5min. A progress curve was then plotted for each fraction to
determine the change in absorbance over time. The sucrose isolation media was used as a blank.

1. Quantification of ATP using Absorption by Ultraviolet Light:
Absorption of ultraviolet light by ATP is a function of the adenine group. The given molar extinction
coefficient of ATP at pH 7 at 295nm is. The optical density of sample A was determined to be
0.664nm at an ABS of 295nm:
Therefore the Beer-Lamberts law states:
Where A is the absorbance, is the molar extinction coefficient, c is the concentration of the sample
and is the path length (i.e. distance travelled by the light through the cuvette).
Therefore sample A containedof ATP, however the sample pH was higher and did not correlate to the
pH of 7 that allowed for the assumption of a molar extinction coefficient of.
Fig. 2.1: The absorbance spectrum of unknown ATP sample A ranging from 210nm to 290nm. An
absorbance peak of 260nm was observed that correlate to, and therefore confirms the presence of
Fig 2.2: Standard curve for the quantification of ATP using the ribose content. It was assumed that
1mol of ribose amounted to 1mol of ATP. The equation for the best fit line: with a linear regression of
0.901. The unknown ATP content of sample A was determined to be 2.425 mg/ml. Note a 1:100
dilution factor was used.

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* 1:100 dilution factor
In regards to the five point standard curve (fig.2.2), the ribose standard concentrations of 0.05 mg/ml
was omitted. This was due to spectrometry errors as the instruments showed varying absorbencies
in the higher concentrations values which did not correspond to expected results after multiple
replicates. It is believed this was due to instrument damage during renovations to the Nelson
Mandela Metropolitan University (NMMU) biochemistry department.
Fig 2.3: Standard curve for the VIS microtiter plate protein quantification of the unknown sample A,
using the bicinchonic acid (BCA) assay. The equation for the best fit line: with a linear regression of
0.998. The unknown protein content of sample A was therefore determined to be 1.226mg/ml. Note a
1:10 dilution factor was used.
Fig 2.4: Standard curve for the quantification of unknown protein of sample A by means of the
fluorescamine assay. The equation for the best fit line: with a linear regression of 0.977. The
unknown protein content of sample A was therefore determined to be 3.191mg/ml.
In regards to fig2.4 the final point on the curve that amounted to a concentration of 500g/ml, was
omitted so as to increase the correlation coefficient closer to 1.0. This was due to high standard
deviations and emission values which did not correspond to the concentration.
Table 2.1: The ATP and Protein concentration and percentage of unknown sample A

ATP (mg/ml)
Protein (mg/ml)

ATP (%)
Protein (%)
UV Quantification by extinction coefficient


Quantification by Ribose Content



BCA Protein Assay


Fluorescamine Protein Assay


High discrepancies between ATP and protein quantification was observed between the given
techniques, as large percentage variations were evident.
Calculations (e.g. UV Quantification by extinction coefficient):
Fig 2.5: Succinate dehydrogenase (SDH) activity assay indicating the change in absorbance
@600nm as a function of mitochondrial concentration within the given fractions. The following
fractions were assayed: the homogenate (H), pellet 1, 2 and 3 (P1, P2, and P3) and the
supernatants 1, 2, and 3 (S1, S2, and S3).
The greatest decrease in absorbance (fig 2.5) was observed to be the homogenate fraction (H),
followed by the first pellet (P1). P1 was obtained after the centrifugation step at 3000xg for 20min.
The subsequent fractions did not yield high decreases in absorbance as compared to the H and P1
fractions and therefore did not show significant mitochondrial activity.
Fig 2.6: Bar graph representation of the succinate dehydrogenase (SDH) activity assay as seen in
fig 2.5. Bars indicate the change in absorbance @600nm over the initial first minute of the reaction.
Determination of succinate dehydrogenase activity (e.g. Fraction H):
X -1
As observed in fig 2.5 the highest degree of activity was observed for the homogenate fraction (H)
followed by the first pellet (P1). The subsequent fractions did not show high mitochondrial activity in
retrospect and therefore were not significant.


Section A
The quantification of ATP of the unknown sample A by ultraviolet light as a function of Beer-Lamberts
Law showed that 0.219% of the sample comprised of ATP. This was assumed that the conditions for
absorption of ATP was constant and related to previous studies where the molar extinction
coefficient was determined at pH 7. The unknown sample A did not however, have a pH of 7 but one
that was considerably higher which would lead to possible inaccuracies in the quantification. The
altering of the samples environment due to pH inconsistencies results in protonation/deprotonation
of the sample and therefore affects the distribution of electrons within the chromophore. This will
correlate to differences between absorbance spectrums at varying pH values (Sheehan, 2009).
However, when taking the observed absorbance spectrum of the unknown sample A into account
(fig.2.1), there were no significant shifts in the spectrum. This would indicate no true alterations the
structure of the sample occurred due to the incorrect pH value and ill effect on the observed ATP
concentration calculated. The method for pH quantification assumed that the molar extinction
coefficient for ATP was at a wavelength of 295nm, however this is incorrect. A molar extinction
coefficient of this value for ATP correlates to a wavelength of 259nm and not one of 295nm therefore
quantification of the sample was measured incorrectly and cannot be considered accurate (Gerstein,
2004). The absorption spectrum (fig.2.1.) for ATP strengthens this conclusion as an absorbance
peak was observed at 260nm (i.e. very close to 259nm) and not near 295nm. Due to these
inaccuracies the results obtained for the unknown ATP concentration cannot be consider valid and
therefore determination relies on the ATP quantification by a function of ribose content and the
orcinol assay.
The orcinol assay for ribose content yielded results that indicated that 24.25% of the unknown
sample A comprised of ATP. However, these results in addition cannot be considered accurate. The
spectrophotometery instrumentation used produced varying results primarily at the higher
absorbance values at a wavelength of 660nm. It was determined that this was due to decalibration
and/or damage to the instrumentation during renovations to the laboratory where the instruments
were stored. This resulted in the removal of the higher concentration points of the standard curve to
obtain a respectable correlation coefficient (fig.2.2.). For an accurate result in this case the
concentration of the unknown sample is required to fall between the first three
concentration/absorbance values of the standard curve. However, the true amount of damage
sustained by the spectrophotometery instrumentation cannot be fully assessed and therefore the
results obtained should not be considered.
The quantification of ATP of unknown sample A by both methods namely; quantification of ATP by
absorption of UV light and the quantification of ATP by the determination of the ribose content were
not accurate. Therefore, modification of the methods should be carried out whereby the pH is more
stringently controlled and quantification should be carried out at the correct wavelength (i.e. 259nm)
in the case of the first method. In the case of the second method, the accuracy of the instruments
used should be verified prior to use. There are however, other methods to determine the ATP content
of a sample more accurately, one of which is high performance liquid chromatography (HPLC).

Manfredi et al. in their measurements of ATP in mammalian cells optimized a HPLC protocol for the
quantification of ATP, ADP and AMP. With the use of ion-pair reverse-phase HPLC system with a
phosphate-buffered acetonitrile gradient mobile phase the ATP of the unknown sample A can be
quantified (Manfedi et al., 2002). This method will be more effective as contaminates would not
interact and interfere with the ATP due to varying dissociation coefficients allowing for only ATP
Protein determination by the bicinchonic acid assay has been determined to be sensitive to 0.5-10g
protein/ml when performed as a microassay, and is in most cases chosen over the Lowry assay
method as it is more tolerable to interfering compounds (Walker, 1994). Generally the absorbance
used for the BCA assay is 562nm however, in this case, an absorbance of 540nm was used. In
regards to filter based plate readers a wavelength range of 540 to 590nm can be used when
performing the reaction without a large degree of sensitivity loss (Burgess and Deutscher, 2009).
Therefore, any inaccuracies that may have occurred due to the chosen wavelength do not have to
be considered in this case. From this method it was determined that 12.26% of the unknown ATP
sample A contained protein, however an absolute concentration of protein cannot be determined.
This is due to the fact that the BCA assay is dependent on the amino acid composition of the protein,
and therefore discrepancies will arise when using a bovine serum albumin standard curve (BSA).
That is to say the BSA amino acid/peptide composition will differ slightly to that of the unknown
protein sample (Walker, 1994).
Fluorescamine assay for protein determination in comparison to the BCA assay procedure is
considerably more sensitive. Fluorescamine allows for the quantification of proteins at levels as low
as 25-50pmoles (Lawrence and Frei, 2000). It was determined from the fluorescamine assay that
31.91% of the unknown sample A comprised of protein which is over twice that determined by the
BCA assay. The amount of protein present was determined through the 1:100 dilution of the
unknown ATP sample A as fluorescamine has a greater sensitivity at lower concentrations and this
dilution yielded results with the lowest standard deviation. The fluorescamine assay was carried out
at a pH of 7.4 which is not ideal for maximum sensitivity as the low pH can strongly influence the
fluorescamine intensity. Maximum sensitivity occurs at a pH of 9 (Keyes, 2000). For best results the
method should be altered to account for the change in pH and compared to the previous
fluorescamine assay results on the unknown sample A. This will allow for a comparison that will
show whether or not the observed unknown sample A protein concentration was indeed altered by
the low pH. At this stage however, it can be assumed that the protein content of the unknown sample
A was in the region of about 20% when considering both BCA and the fluorescamine assay results.
In conclusion, the determination of the ATP content of the unknown sample A by the two stipulated
methods was unsuccessful. This was due to incorrect absorbance values in the case of ultraviolet
quantification and faulty spectrophotometery instruments. In the case of the protein determination of
unknown sample A by the two stipulated methods discrepancies between the methods arose which
didn't allow for absolute protein quantification. However, a rough estimate could be determined but
would be inadequate for most subsequent applications with the sample.

Section B
Differential centrifugation of liver homogenates for mitochondrial isolation generally follows a very
similar procedure, where the homogenate is first centrifuged at a low centrifugal force to remove cell
debris and nuclei (700xg). The centrifugation of the subsequent supernatant is carried out at a much
higher centrifugal force (e.g. 20000g) to separate most of the mitochondria from the fraction. This
would result in the second pellet having the highest mitochondrial activity in comparison to any
possible fractionation steps carried out (Ghosal and Srivastava, 2009). In regards to the method
used in this experiment the first centrifugation step was performed at a high centrifugal force
resulting in most of the mitochondria present in the first pellet as opposed to the second pellet in the
general isolation techniques. This would account for the indicator enzyme, succinate
dehydrogenases' high observed activity in pellet 1. The homogenate also showed very high
mitochondrial activity as to be expected as it would contain the highest concentration of
mitochondria. However, in relation to pellet 1 the homogenate should not have as high a level of
mitochondrial purity due to the presence of the cell debris and nuclei which would interact and
interfere with enzymatic reactions. The low levels of succinate dehydrogenase activity in the
subsequent fractions after pellet 1 would be an indication of mitochondrial cross contamination
during the stepwise fractionation procedure. That is to say residual mitochondria remained within the
supernatant of pellet 1. This would be due to the relatively low centrifugal force applied to the
homogenate to create the mitochondrial pellet allowing for residual mitochondria to remain in the
For a higher isolation yield of mitochondrial isopycnic centrifugation should have been carried out
allowing for a purer mitochondrial product. The fraction enriched in mitochondria (i.e. pellet 1) would
be subjected to resuspension in a 20% saccharose solution and then placed in a centrifugation tube
which contains a saccharose gradient. The gradient would be required to be 20% saccharose at the
top of the centrifugation tube and 50% saccharose at the bottom. High speed centrifugation will
therefore separate the mitochondria from the lysosomes and peroxisomes as a function of density
yielding higher resolution (Vignais and Vignais, 2010).
In conclusion it was observed that mitochondrial isolation from a chicken liver sample occurs in the
first or second fraction of stepwise differential centrifugation, depending on protocol variations. This
method of isolation is sufficient considering a low level of purity is acceptable for the desired