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