A generalized description of SDS-PAGE and visualization through silver staining.

2.  Sodium dodecyl sulphate polyacrylamide gel
electrophoresis(SDS-PAGE) is an analytical
technique used to separate proteins based on
their molecular weight.
 Electrophoresis is a technique used to
separate macromolecules in an electrical
field.
 This technique employs discontinuous
polyacrylamide gel for support and SDS for
denaturation.

3.  SDS (also called lauryl sulfate) is an anionic
detergent.
 Its molecules have a net negative charge
within a wide pH range.
 A polypeptide chain binds amounts of SDS in
proportion to its relative molecular mass.
 The negative charges on SDS masks all the
charges present on protein molecules giving
the protein a net negative charge.

4. SDS molecule
12 carbon tail attached to a sulphate group

5.  Polyacrylamide is a polymer formed from
acrylamide subunits.
 It is highly water-absorbent, forming a
soft gel when hydrated.
 Polyacrylamide is ideal for protein separations
because it is chemically inert and electrically
neutral.
 It forms a mesh like matrix which allows for
separation based on weight.

6.  When proteins are separated by electrophoresis
through a gel matrix, smaller proteins migrate
faster due to less resistance from the gel matrix.
 SDS is a detergent with a strong protein-
denaturing effect and binds to the protein
backbone in ratio to its mass. In the presence of
SDS and a reducing agent(mercaptoethanol) that
cleaves disulfide bonds critical for proper
folding, proteins unfold into linear chains with
negative charge proportional to the polypeptide
chain length.

7.  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

9.  Gel is polymerized between two glass plates
in a gel caster or casting frame, one is short
plate and the other is spacer plate.
 Clamping frame to hold gel cassettes inside
the tank.
 Electrode assembly.
 Combs for well formation.
 Power supply.

10.  The gel used in SDS may or may not be a
discontinuous system.
 Discontinuous system comprises of a 5% stacking
and 4-15% resolving gel based on the
concentration of TEMED.
 Typical components of these gels are:
o SDS
o APS (ammonium per sulphate)
o TEMED(tetra methyl ethylene diamine)
o Acrylamide bis
o Tris (Molarity varies between resolving and
stacking gels).

11.  Hand cast gels can be made in laboratory by the researchers
themselves.
 Align the short plate and stacker plate such that their
bottoms are at exactly the same level and clamp them into
casting frame.
 Place casting frame upon casting stand.
 As a non standard step, water can be poured in between the
two plates, to check for a leak which can later be removed by
filter paper.
 Add all the ingredients into a beaker except for TEMED.
 When the performer is ready to pour the ingredients between
the two plates only right before then the TEMED should be
added to the rest of ingredients.

12.  Resolving buffer is poured between the plates first and
allowed to stand until solidified.
 Butanol can be added upon it to produce a straight bubble
free layer which can later be removed by filter paper.
 Once resolving buffer solidifies Remove butanol and start
pouring the stacking buffer.
 Fill until the mixture reaches the top of short plate
 Ensure absence of air bubbles and insert comb.
 Allow it to stand until solidified.

13.  The sample loaded on a SDS gel usually contains:
o Laemlli buffer
o DI water
o Protein sample
 Laemlli buffer contains:
o SDS
o  Mercaptoethanol
o Tris Hcl pH 6.8
o Bromophenol blue
o glycerol

14.  Add all the ingredients to a centrifuge
eppendorf.
 Centrifuge for1 Minute for run down.
 Heat on thermal cycler or in Hot water at 95
C for 5 minutes.

15.  Assemble the electrophoresis assembly.
 Place the gel cassette in the electrode
assembly with the short plate facing inside of
the electrode assembly.
 For running a single gel place dummy
plate(buffer dam) on the other end of
electrode assembly.
 Remove the comb from gel cassette.
 Load sample in the well produced by combs
using micropipette tips.

16.  Add running buffer into gel box and fill up to
the mark stating two gels or four gels as per
requirement.
 Also add running buffer in between assembly
holding two gel cassettes.
 Place the cap of gel box aligning the color coded
electrodes(red to red and black to black).
 insert the switch into power supply, select the
desired current or voltage and press run.
 Alllow it to run until the sample reaches bottom
end of the gel.

17. Visualization through
Coomassie stain
Visualization through
Silver staining

18. Coomassie stain Silver stain
 Less sensitive
 Less complicated
 Require less steps
 Stain is poured over
gel and left for almost
90 minutes.
 Later de-staining is
done to reveal protein
bands.
 Highly sensitive
 Complex to perform
 Requires various steps
to produce the final
result.

19.  As the electrophoresis completes gel is removed
from the cassette and is placed in fixative
solution for 20 minutes.
 After this fixative is replaced by water which is
left for 10 minutes
 Water is replaced by fresh water which is again
left for 10 minutes.
 Image developing solution then replaces water,
which takes 15-20 minutes to visualize protein
bands present in sample.
 Stopping solution(5% acetic acid) then replaces
developing solution to stop the reaction.

20.  Highly sensitive method
 Very small amount of sample is required.
 Estimation of protein size.
 Estimation of protein purity
 Comparison of polypeptide composition of
different samples.
 Analysis of number and size of polypeptide
subunits.
 Determination of molecular weight.