SDS-PAGE is a technique used to separate proteins by size. Proteins are first denatured and treated with SDS to give each polypeptide chain a negative charge proportional to its molecular weight. The polypeptide-SDS complexes are then electrophoresed through a polyacrylamide gel, allowing separation based on size. Smaller complexes migrate farther and faster through the gel than larger ones. Bands are then visualized after staining to reveal the protein sizes.

1. SDS-PAGE

2.  Proteins – Electrophoresed in polyacrylamide gels under the conditions which
dissociates the proteins into polypeptides
 Strong anionic detergent- SDS is used in combination with a reducing agent and
heat before subjecting to electrophoresis.
 Denatured polypeptides binds SDS and becomes negatively charged.
 The amount of SDS bound is proportional to the molecular weight of the
polypeptide.
 SDS-Polypeptide complex migrates through polyacrylamide gels in accordance to
the size of the polypeptide.
 ~ 1.4 g of detergent is bound per gram of polypeptide.
 Discontinuous buffer system

3.  The SDS-Polypeptide complex swept by a moving boundary created when an
electric current is passed between the electrodes.
 Stacking gel- high porosity, complexes deposited as thin zone on the surface
of the resolving gel.
 The chloride ions in the sample and stacking gel forms the leading edge of
the moving boundary and the trailing edge is composed of glycine molecules.
 Between the leading and trailing edges of the moving boundary is the zone of
lower conductivity and steeper voltage, which sweeps the polypeptides from
the sample and deposits on the surface of the resolving gel.
 The high pH in the resolving gel ionizes the glycine ions and migrates
through the stacked polypeptides and travel through the resolving gel
immediately behind the chloride ions.

5.  SDS-Polypeptide complex move through the resolving gel in a zone of
uniform voltage and pH and are separated according to size by sieving.
 The effective range of separation depends on the concentration of
polyacrylamide and amount of cross-linking.
 Bisacrylamide forms the cross-links which adds rigidity and tensile strength
and forms pores through which the peptide complex pass.
 Commonly employed ratio bisacrylamide: acrylamide (1:29). As the ratio
increases the pore size decreases.

7. Materials required
 Acrylamide: bis-acrylamide solution (29:1)
 Separating gel buffer (1.5 M Tris, pH 8.8)
 Stacking gel buffer (0.5 M Tris, pH 6.8)
 10% SDS
 10% APS
 TEMED
 Bromophenol blue
 Sample buffer
 Tank buffer (Tris-Glycine)
 Staining solution (CBB)
 Destaining solution

8. Procedure
 Clean the glass plate thoroughly.
 Place the notched plate on the paper and place the spacers strips on both sides
and at the bottom.
 Mix the separating gel solutions gently.
 Add SDS and APS into the solution and pour the solution into the glass plate
using a pipette up to the level of 4 cm from the top.
 Using a pipetman, layer carefully on the gel with the layering solution (n-
butanol or milli Q). Layering helps to form a smooth, even gel surface and also
excludes oxygen on the surface (as oxygen inhibits gel polymerization)
 Allow the gel to stand for 30 min to polymerize.

9.  Once the gel is polymerized, pour off the top layer by tilting the gel, wash gel
surface gently with distilled water, wipe dry the surface and inside of the glass
plate with Whatman No.1 filter paper strips.
 Mix the gel solutions gently for stacking gels as mentioned above.
 Fill the top of the gel with stacking solution and insert the comb gently between
the gel plates.
 Allow the gel to stand for 30 min to polymerize. Rinse the wells with distilled
water and invert it to drain the wells.
 Fill the upper and lower chambers with running buffer. Switch on the power
supply.
 Prepare the sample solutions by mixing (1:1) in 2x sample buffer and placing the
tubes for 7 ml in a boiling water bath. Cool to room temp, spin for 1 min and load
using micropipettes.

10.  Load the protein molecular weight standards in one of the wells.
 Electrophorese the gel at 80 volts until the tracking dye has reached 0.5 cm
from the bottom of the gel
 Turn-off the power supply, disconnect the leads and remove the glass plates
from the tank.
 Gently slide the gel into a tray containing the dye solution.
 Stain the gel for 2-3 hours or overnight at room temperature.
 Pipette out the staining solution and replace with the destaining solution.
Change the destaining solution 2 -3 times.