Biochemistry Laboratory (BCM 362L), Experiment No.

5, © February 14, 2006

3nd Quarter A.Y. 2005-2006

Gel Electrophoresis

Mr. *****1, *****, *****2

1
Professor, School of CHE-Chm, Mapua Institute of Technology
2
Student, BCM 362L/ A31, School of CHE-Chm, Mapua Institute of Technology
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ABSTRACT

Sodium Dodecylsulfate Polyacrylamide Gel buffer system that incorporates SDS was also
Electrophoresis is a procedure used for used. Casein, albumin and standard proteins
separating a mixture of molecules through a were analyzed and their molecular weight was
stationary material in an electrical field. In this measured by extrapolating using their Rf values.
case the stationary material used is the
polyacrylamide gel. The Mini-PROTEAN 3 Keywords: electrophoresis, buffer, gel,
module was used in this experiment. The polyacrylamide.
Laemmli buffer system which is a discontinuous
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INTRODUCTION the level of the comb. The following stock

Gel Electrophoresis is a method that
separates macromolecules either nucleic acids or
proteins on the basis of size, electric charge, and
other physical properties.
A Gel is a colloid in a solid form. The
term Electrophoresis describes the migration of
charged particle under the influence of an
electric field. Electro refers to the energy of
electricity. Phoresis, from the Greek verb
phoros, means "to carry across." Thus, Gel
electrophoresis refers to the technique in which
molecules are forced across a span of gel,
motivated by an electrical current. Activated
electrodes at either end of the Gel provide the
driving force. A molecule's properties determine
how rapidly an electric field can move the
molecule through a gelatinous medium.
Many important biological molecules
such as amino acids, peptides, proteins,
nucleotides, and nucleic acids, posses ionisable
groups and, therefore, at any given pH, exist in
solution as electrically charged species either as
cations (+) or anions (-). Depending on the
nature of the net charge, the charged particles
will migrate either to the cathode or to the
anode.3
METHODOLOGY
solutions and buffers were prepared:
1. Acrylamide/Bis (39%, 2.67% C)
87 g of acrylamide (29.2 g/100 mL) was
mixed with 2.4 g N’N’-bis-methylene-
acrylamide (0.8g/100mL). It was diluted with
300 mL deionized water.
2. 10% (w/v) SDS
10 g of SDS was dissolved in 90 mL of
water with gentle stirring and was diluted to 100
mL with deionized water.
3. 1.5 M Tris – HCl, pH 8.8
27.23 g of Tris base (18.15/100 mL)
was added to 80 mL deionized water. It was
adjusted to pH 8.8 with 6 N HCl. The total
volume was brought to 150 mL with deionized
water and was stored at 4ºC.
4. 0.5 M Tris – HCl, pH 6.8
6 g of Tris base was added to 60 mL
deionized water. It was adjusted to pH 6.8 with 6
N HCl. The total volume was brought to 100 mL
with deionized water and was stored at 4ºC.
5. 10% APS
100 mg of ammonium persulfate was
dissolved in 1 mL of deionized water.

The gel apparatus was set-up. A mark The 10% gel was prepared by mixing
was placed on the gas plate which is 1 cm below 4.1 mL DDI H2O, 3.3 mL 30% Degassed
Acrylamide/Bis, 2.5 mL Gel buffer (1.5 M Tris-
HCl for resolving gel buffer; 0.5 M Tris-HCl for
stacking gel buffer), and 0.1 mL 10% w/v SDS.
The resolving gel was mixed with 50 µl
10% APS and 5 µl TEMED and was
immediately poured on the comb. After 20
minutes water was overlayed on the gel for a few
minutes. The water was then discarded and the
Stacking Gel was added and a comb was placed
on the layer for the formation of twelve wells.
The proteins were then added to these wells.
After several minutes the gel is placed
on the apparatus for electophoresis in 40
minutes. The staining solution was prepared by
mixing 50 mL methanol, 10 mL glacial acetic
acid and 0.25 mg Coomasie brilliant blue, and
was diluted to 100 mL. The gel was removed for
the glass plates using a metal spatula and was
incubated in the staining solution until the
desired band intensity was achieved. It was then
washed with a destaining solution which
contains 25 mL 95% ethanol and 5 mL glacial
acetic acid diluted to 100 mL of distilled
deionized water. The gel was dried for analysis.
RESULTS AND DISCUSSION
The support matrix used was
polyacrylamide which is a means of separating
molecules by size, in that they are porous gels.
The porous gel acts as a sieve by retarding, or in
some cases completely obstructing, the
movement of large macromolecules while
allowing smaller molecules to migrate freely.
Polyacrylamide, which is easy to handle and to
make at higher concentrations, is used here to
separate casein, albumin and other proteins and
small oligonucleotides that require a small gel
pore size for retardation.
SDS is an anionic detergent which
denatures proteins by "wrapping around" the
currents caused by small temperature gradients,
and it minimizes protein movements other than
those induced by the electric field. Proteins can
be visualized after electrophoresis by treating the
Gel with a stain such as Coomassie blue, which
binds to the proteins but not to the Gel itself.
Each band on the Gel represents a different
protein (or a protein subunit); smaller proteins
are found near the bottom of the gel.
A linear relationship exists between the
logarithm of the molecular weight of an SDS-
denatured polypeptide, and the Rf. Rf is
calculated as the ratio of the distance migrated
by the molecule to that migrated by a marker
dye-front. The values calculated are located
below:
Lane 1 2 3 7 8 9
0.746 0.481 0.730 0.42 0.437
0.625
6 0 8 5 5
Rf
0.759 0.72
0.75
5 5
The lanes contain casein, albumin and
standard protein. Those with almost similar
values can assumed to be the same protein
because of the close similarities of their
molecular weights (Lane 1,3; 2,7,8; 9). The bluer
the band, the higher is the concentration of the
protein. Lanes that is not recorded may contain
bands; hence the authors may have errors on not
locating it. A simple way of determining relative
molecular weight by electrophoresis is to plot a
standard curve of distance migrated vs. log MW
for standard samples (220,000; 170,000;
116,000; 76,000; 53,000 Da), and read off the
log MW of the sample after measuring distance
migrated on the same gel.
5.4
5.3
5.2

polypeptide backbone - and SDS binds to the 5.1

log MW

5
proteins fairly specifically in a mass ratio of 4.9
1.4:1. In so doing, SDS confers a negative 4.8
charge to the polypeptide in proportion to its 4.7

length - ie: the denatured polypeptides become 4.6

0 0.2 0.4 0.6 0.8
"rods" of negative charge cloud with equal Rf
charge or charge densities per unit length. It is
usually necessary to reduce disulphide bridges in
proteins before they adopt the random-coil
configuration necessary for separation by size:
this is done with 2- mercaptoethanol. CONCLUSION
Different samples are loaded in wells or
depressions at the top of the polyacrylamide gel. Electrophoresis is the migration of
The proteins move into the Gel when an electric charged molecules in solution in response to an
field is applied. The Gel minimizes convection electric field. Their rate of migration depends on
the strength of the field; on the net charge, size
and shape of the molecules and also on the ionic
strength, viscosity and temperature of the
medium in which the molecules are moving. As
an analytical tool, electrophoresis is simple,
rapid and highly sensitive. It is used analytically
to study the properties of a single charged
species, and as a separation technique.
The lower the band, the higher the
molecular weight of the protein, and vice versa.
The resolution obtained in a
discontinuous system is much greater than that
obtained with a continuous system.
Polyacrylamide gels offer greater
flexibility and more sharply defined banding
than agarose gels.

LITERATURE CITED

1. Biochemistry, Garrett and Grisham, 3rd

Edition

2. © 1993-2003 Microsoft Corporation. All

rights reserved.

3.http://www.bergen.org/AAST/Projects/Gel/intr
o.htm

4.http://www.uct.ac.za/microbiology/sdspage.ht
ml