SCIENTIFIC RESEARCH FOR BIOCHEM LAB

ISOLATION AND CHARACTERIZATION OF BIOLOGICAL MACROMOLECULES USING

FILTRATION AND CENTRIFUGATION

Joebeth C. Competente

University of Santo Tomas- Legazpi

ABSTRACT
 INTRODUCTION

The extraction of biomolecules, DNA, RNA, lipids and protein, is the most crucial method
used in molecular biology. It is the starting point for downstream processes and product
development including diagnostic kits. DNA, RNA, and protein can be isolated from any biological
material such as living or conserved tissues, cells, virus particles, or other samples for analytical or
preparative purposes. (M.Wink, 2006).Over the years, a number of sophisticated analytical and
preparative techniques have been developed for separating, analyzing, and isolating the various
macromolecular constituents of cells and tissues. Various forms of electrophoresis,
chromatography, and ultracentrifugation are now in routine use in most lab-oratories engaged in
molecular biological studies and have greatly increased our understanding of the chemistry and
properties of the cellular macromolecules (Anandita, 2007). Most of the methods that are used to
separate and iso-late different members of a class of macromolecules simultaneously provide
information concerning their chemistry because parameters such as molecular size, shape, density,
net and absolute charge, differential solubility, and so forth are used as the basis for the separation
(Piradyha, 2002).

Biomolecules are those which are produced by the living organisms and are very important
for their day to day activities. They are structurally dependant and lose their function on disruption.
They are usually composed of simple subunits called monomers, which combine chemically to
form large and complex polymers. Carbon, Hydrogen and Oxygen are the common elements which
make all of the biomolecules. Biomolecules are broadly classified into four categories, like
carbohydrates, proteins, lipids and nucleic acids. (Maria, 2017)

Proteins is an essential constituent of all cells. Proteins are polymers of amino acids. There
are 20 naturally occurring amino acids commonly found in proteins. The sequence and organization
of these amino acids contribute in the unique physico-chemical characteristics of a protein which,
in turn, determine what protein isolation and purification technique should be used. These
characteristics include molecular structure, molecular weight, solubility in different solvents,
isoelectric pH, and heat stability (Bathan et.al, 2017). In the eighteenth century, proteins were
known as a distinct class of biological molecules by Antoine Fourcroy and others. They
distinguished this molecule by its ability to coagulate under treatment with heat or acid. However,
the first description of protein was carried out by Gerhardus Johannes Mulder, a Dutch chemist, in
1893. His studies on the composition of animal substances, mainly fibrin, albumin, and gelatin,
showed the presence of carbon, hydrogen, oxygen, and nitrogen . Furthermore, he recognized that
sulfur and phosphorus were present sometimes in animal substances that consisted large number of
atoms and he established that these “substances” were macromolecules. Most of the early studies
focused on proteins that could be purified in large quantities. For example, blood, egg white and
various toxins. Most of the proteins are hard to purify in more than milligram quantities even with
today’s highly advanced methods. A majority of techniques for protein purification were developed
in a project led by Edwin Joseph Cohn, a protein scientist, during World War II. He was responsible
for purifying blood and worked out the techniques for isolating the serum albumin fraction of blood
plasma, which is important in maintaining the osmotic pressure in the blood vessels, which help
keep soldier alive (D. Whitford, 2005).

Nucleic Acid is a naturally occurring chemical compound that is capable of being broken
down to yield phosphoric acid, sugar and a mixture of organic bases (purines and pyrimidines). It
is the main information-carrying molecules of the cell, and by directing the process of protein
synthesis, they determine the inherited characteristics of every living thing. The two main classes
of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the master
blueprint for life and constitutes the genetic material in all free-living organisms and most viruses.
RNA is the genetic material of certain viruses, but it is also found in all living cells, where it plays
an important role in certain processes such as the making of proteins (Roberts, 2007). The very first
DNA isolation was done by a Swiss physician, Friedrich Miescher in 1869. He hoped to solve the
fundamental principles of life, to determine the chemical composition of cells. He tried to isolate
cells from lymph nodes for his experiment but the purity of lymphocytes was hard and impossible
to be obtained in sufficient quantities. Therefore, he switched to leucocytes, where he obtained
them from the pus on collected surgical bandages. Initially, Miescher focused on the various type
of protein that make up the leukocytes and showed that proteins were the main components of the
cell’s cytoplasm. During his tests, he noticed that a substance precipitated from the solution when
acid was added and dissolved again when alkali was added. This was, for the first time he had
obtained a crude precipitate of DNA. To separate DNA from the proteins in his cell extracts,
Miescher developed new protocol to separate the cells’ nuclei from cytoplasm and then isolated
DNA. However, his first protocol failed to yield enough material to continue with further analysis.
He had to develop a second protocol to obtain larger quantities of purified nuclein, which had been
named as ‘nucleic acid’ later by his student, Richard Altman (R. Dahm, 2004).

Carbohydrates are divided into three general classes depending on monosaccharide units
they contain. Monosaccharide or simple sugars are further classified by the number of carbon atoms
they contain and it cannot by hydrolyzed. Oligosaccharide are subdivided into disaccharide,
trisaccharides and so forth. Polysaccharide can be made up of hundreds of thousands of
monosaccharide units connected in various patterns. (Loffredo, W. M. 2002) Lipids are a
heterogeneous group of compounds synthesized by living cells and generally soluble in non-polar
organic solvents and insoluble in water. Differences in the solubility of the organic solvents are
essential in the separation if lipids from other biomolecules such as carbohydrates and proteins.
(Barreto, M. C. 2005)

This study aimed to obtain quantitative and qualitative results- its composition,
identification and characterization through filtration and centrifugation during the isolation or
extraction process.

Method

ISOLATION AND CHARACTERIZATION OF PROTEINS

Isolation of Proteins

Exp. #1 Casein from Skimmed Milk

Materials

Powdered, non-fat milk 20.0 g

10% CH3COOH

Thermometer

pH meter/indicator

Funnel and filter paper

Hot plate

Procedure

Place 20.0 g of powdered, non-fat milk and 50.0 mL of water into a 100 mL beaker, should
mix well. Heat the mixture to about 40 oC, monitor the temperature with a thermometer and not let
it exceed to 40 oC. When the mixture reaches 40 oC, add 10% CH3COOH drop wise. Stir the
solution gently after 5 drops. Continue adding acetic acid until the pH reaches 4.6. filter off the
congealed casein by gravity (or vacuum) filtration. Set aside the filtrate for the isolation and
quantitative analysis of albumin. Dry the casein residue and calculate the weight % of casein
isolated from the powdered milk.
Exp #2 Albumin from Skimmed Milk

Materials

Filtrate from the isolation of casein

Thermometer

Hot Plate

Procedures

Transfer half of the filtrate in a small beaker. Set aside the remaining half for quantitative
protein analysis. Heat the filtrate in the beaker to 75 oC for about 5 minutes using a hot plate and
decant off the liquid from the precipitated albumin.

Exp. #3 Gluten from Wheat Flour

Materials

1 cup of wheat flour

Cheesecloth

0.01 M iodine solution

Procedure

Add water to one cup of wheat flour to make a thick dough. Wrap the dough with the
cheesecloth and place it under running water until all the starch is removed. Test the dough by
washing it with iodine solution until a negative result is obtained. Collect the gluten (the insolutable
material) for hydrolysis and qualitative protein analysis.

Exp. #4 Myoglobin from Muscle

Materials

6.0 g of Minced Beef

Cheesecloth

Centrifuge

Centrifuge tubes
 (NH4)2SO4 Crystals

70% buffer-diluted (NH4)2SO4 solution, pH 7.5

Procedure

Place 6.0 g of the minced beef and 6 ml of the 70% (NH4)2SO4 solution in a small beaker. Gently
stir the mixture for 1 minute to release the myoglobin. Express the dark-red extract into a new
beaker using cheesecloth and centrifuge the extract at 13,000 x g for 5 minutes. Transfer 1.5 mL of
the supernatant into another empty centrifuge tube. Add ~0.3-0.35 g of the (NH4)2SO4 crystals,
ground to fine powder. Mix gently until dissolves and avoid it from frothing. Centrifuge the sample
again for at least 5 minutes and decant off the supernatant.

ISOLATION AND CHARACTERIZATION OF NUCLEIC ACID

Materials

Deoxyribonucleic Acid (DNA)

Table 1. Reagents and Materials for Isolation

Plant Isolation Onion Isolation Animal Isolation

 Freshly harvested young  2 yellow onions  Chicken liver
papaya/other leaves  Homogenizing  Lysing buffer:
 Cetyltrimenthylammonium solution: 5% SDS  0.5 M Tris-HCl,
bromide (CTAB) (sodium dodecyl
pH 8.0
 1 M Tris-HCI, pH 8.0 sulfate or sodium
 0.5 M EDTA
 0.5 M EDTA lauryl sulfate)
 0.15 M NaCl  5 M NaCl
 5 M NaCl
 Chloroform: Isoamyl  0.15 M  5% SDS NaClO4
Alcohol (24:1) Na3C6H5O7  Chloroform:
 95% ethanol (sodium citrate) isoamyl alcohol
 70% ethanol  0.01 M EDTA
(24:1)
 Commercial papain
 Ice-Cold 95%
or meat tenderizer
(6% in water) ethanol
 Ice-cold 95% ethanol  70% ethanol

Procedures
Plant DNA Isolation

This experiment will be conducted with usage a CTAB Method. It is composed of 100 mL: 2 g of
the CTAB; 10 mL of the 1 M Tris-HCl, ph 8; 4 mL of the 5.0 M EDTA; 26 mL of the 5 M NaCl;
and 58 mL of the distilled water. Pre-heat the buffer at 60 oC. Harvest 10-15 g young papaya leaves,
wash it with tap water and rinse it off with distilled water. Blot-dry with a towel and cut off the
large veins. Pack the leaves in dry ice until frozen or thoroughly chilled. Grind the frozen leaves in
a chilled mortar and pestle and transfer the sample in a chilled 50 mL test tube. Immediately add
15 mL of the pre-heated CTAB buffer and mix gently. Place the mixture in a 60 oC water bath for
30 minutes and gently swirl occasionally. Centrifuge the mixture at 16,000 x g for 15 minutes at
room temperature and transfer the supernatant to a clean test tube. Add one volume of chloroform:
isoamyl alcohol and mix gently. Centrifuge the mixture again with the same time and speed?????
And transfer the upper aqueous layer using a wide-bore pipette to a new 50-mL test tube. Add two
volumes of the ice-cold 95% ethanol by carefully pouring the ethanol down the inner wall of the
test tube. Mix gently to precipitate the nucleic acids and spool out the DNA that precipitates out of
the solution using a glass rod. Transfer it to another tube and resuspend it in the TE buffer.
Centrifuge at 5,000 x g at room temperature for 10 minutes and discard the supernatant. Add 5-10
mL of the 70% ethanol to the pellet and centrifuge it again with another 5,000 x g at room
temperature with the same range of time. Pour off the supernatant and resuspend the DNA pellet in
3-5 mL of the TE buffer.

DNA Isolation from an Onion

Place 50 mL of the homogenizing solution in a 125-mL Erlenmeyer flask and heat it in a water bath
until the solution reaches 60 oC. Add 25 g minced onion to the pre-heated homogenizing solution.
Stir and let the solution sit it in the water bath for 5 minutes. Put 1.5 g of the papain (or 4 mL of the
meat tenderizer solution) and keep the solution in the 60 oC Water bath for 10 more minutes.
Immediately place the flask in an ice bath for 5 minutes, swirl the solution gently. Quickly pour the
contents of the flask into a blender and homogenize them for 45 seconds. Filter the homogenate
through 4 layers of cheesecloth into a 250 mL beaker and cool the solution on ice. Using a pipette,
immediately add 15-20 mL of the ice cold ethanol, allowing it to slowly drip down the side of the
tube. There should be a clear layer of ethanol formed on top of the onion filtrate. When ethanol is
added to the mixture, all the components of the mixture (except for the DNA) will stay in the
solution and DNA will precipitate out. Let the tube sit for 3-5 minutes without being disturbed,
DNA will become visible as white strings in the ethanol layer. Leaving the solution overnight in
the refrigerator is highly recommended. Spool the DNA by snagging it with a Pasteur pipette whose
hook is bent on the tip. Transfer the DNA to a clean test tube and resuspend it in the TE buffer or
SSE solution.

Animal DNA Solution

To perform this experiment the lysing buffer must be pre-heated in a 60 oC water bath. Pack the
fresh chicken liver in dry ice until thoroughly chilled and homogenize 25 g of it in a blender with
enough ice-cold distilled water to make a uniform suspension. Transfer the homogenate to a pre-
chilled Erlenmeyer flask and immediately add enough pre-heated lysing buffer; mix it gently. Place
the mixture in a 60 oC water bath for 30 minutes and gently swirl occasionally. Add 10 mL of the
5 M NaClO4 and place the mixture back in the 60 oC water bath for another 10 minutes. Centrifuge
at 13,000 rpm for 10 minutes. Collect the supernate and add an equal volume of the chloroform:
isoamyl alcohol. Mix gently and centrifuge it again with the same speed in 5 minutes. Three layers
should be visible: a bottom organic phase, a middle band of denatured protein at the interface and
an upper aqueous layer containing the nucleic acids. With a use of a wide-bore pipette, carefully
pipet the upper aqueous phase and put it in a clean test tube. Add 2 volumes of ice-cold 95%
ethanol. Mix gently with a glass rod and spool the DNA precipitate. Wash it twice with enough
70% ethanol and resuspend the precipitate in the SSC TE buffer.

Ribonucleic Acid (RNA)

ISOLATION AND CHACRACTERIZATION OF CARBOHYDRATES

Materials

Extraction of Glycogen from Chicken Liver

0.1% Acetic Acid

Distilled water

Bunsen burner

Mortar and pestle

Test tube
Beaker

Hard glass test tube

Procedures

In performing this experiment, the chicken liver must weigh 3 g and be placed on a petri dish.
Mince the sample using a pair of scissors. Pour 12 mL of boiling water on it and stir with a glass
rod. Transfer the mixture into a mortar, grind it thoroughly and then add 3 mL of distilled water
and transfer the mixture into the beaker. Heat it in a boiling water bath for at least 30 minutes, if
necessary add water to the mixture to keep it from drying. Glycogen goes to the solution during
heating and add 1 mL of the 0.1% acetic acid to improve the precipitation of proteins. Filter the
mixture and divide the obtained glycogen solution into a 4 portions into 4 test tubes that are
marked form “1”-“4”. Use the solution in the glycogen precipitation by ethanol experiment,
general tests for polysaccharides and hydrolysis of polysaccharides.

METHODS
PROCEDURE

DATA ANALYSIS

RESULTS AND DISCUSSION