Running of SDS-PAGE Practical

Sepration of proteins on the basis of molecular weight.
SDS is used as denaturing agent along with Polyacrylamide gel.

1. Practical # 14
29-1-2019
RUNNING OF SDS-PAGE
The separation of macromolecules in an electric field is
called electrophoresis. A very common method for
separating proteins by electrophoresis uses a discontinuous
polyacrylamide gel as a support medium and sodium
dodecyl sulfate (SDS) to denature the proteins. The method
is called sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). SDS (also called lauryl
sulfate) is an anionic detergent, meaning that when
dissolved its molecules have a net negative charge within a
wide pH range. A polypeptide chain binds amounts of SDS
in proportion to its relative molecuar mass. The negative
charges on SDS destroy most of the complex structure of
proteins and are strongly attracted toward an anode
(positively-charged electrode) in an electric field.
Polyacrylamide gels restrain larger molecules from
migrating as fast as smaller molecules. Because the charge-
to-mass ratio is nearly the same among SDS-denatured
polypeptides, the final separation of proteins is dependent
almost entirely on the differences in relative molecular
mass of polypeptides. Protein separation by SDS-PAGE
can be used to estimate relative molecular mass, to
determine the relative abundance of major proteins in a
sample, and to determine the distribution of proteins

2. among fractions. The purity of protein samples can be
assessed and the progress of a fractionation or purification
procedure can be followed. Different staining methods can
be used to detect rare proteins and to learn something about
their biochemical properties. Specialized techniques such
as Western blotting, two-dimensional electrophoresis, and
peptide mapping can be used to detect extremely scarce
gene products, to find similarities among them, and to
detect and separate isoenzymes of proteins
Materials:
 To Pour Gels
30% acrylamide 10% SDS 10% Ammonium
persulphate(APS) (make fresh each time) TEMED ((N, N,
N', N'-tetramethylethylenediamine) 1.5 M Tris, pH 8.8
(resolving gel) 1.0 M Tris, pH 6.8 (stacking gel)
 5x SDS Running Buffer (1 L)
Tris 15 g Glycine 72 g SDS 5 g
 Coomassie Blue Stain
10% (v/v) acetic acid 0.006% (w/v) Coomassie Blue
dye 90% ddH2O
 Isopropanol Fixing Solution
10% (v/v) acetic acid 25% (v/v) isopropanol 65%
ddH2O

3.  SDS sample loading buffer (40 ml)
ddH2O 16 ml 0.5 M Tris, pH 6.8 5 ml 50% Glycerol 8
ml 10% SDS 8 ml 2-βmercaptoethanol 2 ml (add
immediately before use), bromophenol.
Procedure:
1. Preparation Of Gels:
Resolving Gel:
1. Combine all reagents except the TEMED (N, N, N', N'-
tetramethylethylenediamine) for the 15% separating gel.
When ready to pour the gel, quickly add the TEMED
0.004mL TEMED.
2. Mix using a Pasteur pipette, and transfer the separating
gel solution between the glass plates in the casting
chamber to about 3/4 inch below the short plate.
3. Add a small layer of absolute ethyl alcohol on top of
the gel prior to polymerization to straighten the level of
the gel and remove unwanted air bubbles that may be
present. Once the gel has polymerized, the ethyl alcohol
can be removed by absorption with filter paper.
4. Dry ethanol at RT to pouring the stacking gel.
Stacking Gel: The stacking gel concentrates proteins
loaded into the sample wells so that they are resolved as a
unified "line" once they enter the stacking gel. The reason

4. for the lower pH is that this "lower ionic strength implies
higher electrical resistance and consequently a higher
electric field, provoking the faster movement of the
proteins and of every other charged particle in the gel
1.Combine all reagents except the TEMED for the 5%
stacking gel.
2.Mix using a Pasteur pipette, and transfer the stacking
gel solution between the glass plates in the casting
chamber.
3.Insert the well forming comb into the opening between
the glass plates.Both the resolving and stacking gels
should polymerize within six minutes.
4.Once the stacking gel has polymerized, the comb can be
gently removed. The polymerized gel between the short
plate and spacer plate forms the "gel cassette".

2. Sample Preparation:
ADD Sample along with the sample loading buffer as
mentioned in the materials.In the buffer SDS is an
anionic reagent which gives negative charge to the
sample by binding with the amino acids,it also breaks
the covalent bonds in proteins. Βmercaptoethanol is a
reducing agent it breaks the sulphide bonds in the
protein thereby giving proteins a primary structures

5. so that shape and charge donot play role in separation
but only the size.Bromophenol is a staining dye it
gives blue color to the sample so that they are visible
on the gel.glycerol will add viscosity so that the
sample donot float in the buffers and move on the
gel.

3. Electrophoresis:
Remove the gel cassette from the casting stand and
place it in the electrode assembly with the short plate
on the inside. Slide the electrode assembly (with the
gel cassette) into the clamping frame. Press down on
the electrode assembly while clamping the frame to
secure the electrode assembly.Pour some 1X
electrophoresis buffer into the opening of the casting
frame between the gel cassettes. Add enough 1X
electrophoresis buffer to fill the wells of the gel. Use a
gel loading tip to pipette some buffer into each well to
ensure cleanliness. When all wells are sufficiently
cleaned, slowly pipette 20µL of denatured sample or
MW marker into each well. When the gel has been
loaded, cover the tank with lid and connect to the
power supply at 80V until the dye front reaches the
bottom of stacking gel.

6. 4. Staining the Gel:
Removal the gel from the cassette. The plates are
separated and the gel is dropped into a staining dish
containing deionized water. After a quick rinse, the water
is poured off and stain added. Staining usually requires
incubation overnight, with agitation.0.1% Coomassie
Blue dye in 50% methanol, 10% glacial acetic acid.
Acidified methanol precipitates the proteins. Staining is
usually done overnight with agitation..The dye actually
penetrates the entire gel, however it only sticks
permanently to the proteins. Excess dye is washed out by
'destaining' with Isopropanol fixing Solution. Properly
stained/destained gels should display a pattern of blue
protein bands against a clear background. The gels can
be dried down or photographed for later analysis and
documentation.
5.Gel Documentation:

7. The gels are observed in the UV OR gel Documentation
to see the bands and are compared with the standard or
marker to find exact molecular weight of the proteins in
the sample.