Sodium Dodeyl Sulfate- Poly Acrylamide Gel Electrophoresis (SDS-PAGE)

Aim:
To separate viral proteins through 10% reducing SDS-PAGE
Principle:
Proteins are reacted with anionic detergent SDS to form negatively charged complexes. Initial
heating of the protein sample at 95ºC in the presence of excess SDS and thiol reagent (2- β- mercapto
ethanol) denatures the protein mixture and disrupts the disulphide bonds. Under these conditions, all
reduced polypeptides bind the same amount of SDS on a weight basis (1.4gm of SDS per gram of
polypeptide) independent of amino acid composition and sequence. The resolving power of SDS-
PAGE is greatly enhanced by preceding the protein separation phase with a “Stacking gel” which
employs the principles of isotachophoresis to concentrate samples from relatively large volumes into
very small bands (or Zones) but does not separate them. In the separating gel, the negatively charged
SDS- protein complexes migrate through the sieve like polyacrylamide matrix and are separated
solely on molecular weight differences.
Materials required:
1. Mini gel Apparatus (Vertical)
2. D.C power supply with Cord
3. Test tubes
4. Eppendorfs
5. Micropipettes and tips
6. Gel storage bag
Reagents required:
1. Acrylamide stock (30%)
2. Separating Gel Buffer (1.5M Tris, pH 8.8)
3. Stacking gel Buffer (1M Tris, pH 6.8)
4. Sodium Dodecyl Sulphate (10% SDS)
5. Ammonium per sulfate (10% APS)
6. N, N, N’, N’- tetra methyl ethylene diamine (TEMED)
7. Sample loading buffer (5X)
8. Electrophoresis buffer
9. Staining Solution
10. Destaining Solution
Preparation of reagents:
1. Acrylamide stock (30%)
Acrylamide 29.2gm
Bisacrylamide 0.8gm
 Distilled water 100ml
Dissolve the salts in 100 ml water

2. Separating Gel Buffer (1.5M Tris, pH 8.8)

Weigh 18.17gm of Tris in 90 ml of distilled water adjust the pH was adjusted to 8.8
using 1N hydrochloric acid and final volume make upto 100 ml
3. Stacking gel Buffer (1M Tris, pH 6.8)
Weigh 12.14gm of Tris in 90 ml of distilled water adjust the pH was adjusted to 6.8
using 1N hydrochloric acid and final volume make upto 100 ml
4. Sodium Dodecyl Sulphate (10% SDS)
Weigh 1 g of SDS and dissolve in10ml of distilled water
5. Ammonium per sulfate (10% APS)
Weigh 1gm of APS and dissolve in10ml of distilled water
6. Sample loading buffer (5X) – Reducing buffer
1M Tris HCl (pH 6.8) 1ml
10% SDS 0.8ml
β- mercapto ethanol 2ml
Glycerol 4ml
Distilled water 2.2ml
Bromophenol blue 100mg
7. Electrophoresis tank buffer (10X)
Tris 15.1gm
Glycine 72gm
SDS 5gm
Distilled water 500ml
Dissolve the salts in 500 ml distilled water. For preparing 1X tank buffer Take 30ml
of 10X Electrophoresis tank buffer and make up to 300 ml with MilliQ Water.(do not
adjust the pH)
10. Staining Solution
0.5% Coomassie Brilliant Blue (R-250) in Methanol: Acetic acid: Distilled Water in
the ratio of 4.5:1:4.5.
11. Destaining Solution
Methanol: Acetic acid: Distilled water in the ratio of 4.5:1:4.5.

Methodology
1. Clean the glass plates by soaking in a detergent and wash thoroughly with water and keep
in hot air oven.
 2. Assemble the plates with spacers using Vaseline and clamp it with the help of clips.
3. Prepare the separating gel mixture as shown in the table and immediately pour into the
assembled plate and allow it to polymerize for 20- 30min.
4. Prepare the stacking gel mixture was as shown in the table and immediately pour on top
of the polymerized separating gel.
5. Insert the comb gently in between the glass plates on the top of stacking gel mixture.
6. Allow the gel to get polymerized for at least 20 min.
7. Preparation of Samples: Prepare the sample solution to be loaded into the wells by
adding the required volume of sample buffer (4 parts of sample and 1 part of sample
buffer). The sample was incubated at 90ºC in a boiling water bath for 5 minutes.
8. When the polymerization of the stacking gel gets over, remove the comb and the lower
spacer strip carefully.
9. Remove excess vacuum grease from the bottom of the gel by wiping with a piece of
tissue paper.
10. Remove the comb carefully and clean the wells using filter paper.
11. Fill the lower reservoir and upper reservoir of the electrophoresis apparatus with the
required volume of tank buffer.
12. Fix the gel plate to the electrophoresis tank carefully with appropriate clips and clamps.
13. Load the protein samples in the wells and equalize with the level electrophoresis buffer in
the upper chamber by filling with electrophoresis buffer.
14. Raise the level of buffer in the upper reservoir.
15. Connect the electrodes to the power pack and the switch on the current and observe for
the formation of bubbles which indicates the passage of current
16. Keep 10-15 mA /50-60 V for stacking gel and 15-20mA/75-100V for separating gel.
17. Turn off the power supply when the tracking dye reached the bottom of the gel and
transfer the gel to the staining solution for 1-2 hours.
18. Later transfer the gel to destaining solution until the clear bands got visible.
19. For Blotting analysis Staining/destaining process will not perform.

SDS PAGE 10% Composition

REAGENTS SEPARATING GEL (10ml) STACKING GEL (5ml)

Distilled water 4.0 3.4
30% Acrylamide 3.3 0.83
1.5M Tris (pH 8.8) 2.5 -
1M Tris (pH 6.8) - 0.63
 10% SDS 0.1 0.05
10% APS 0.1 0.05
TEMED 0.004 0.005

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of

bacteriophage T4. Nature 227(5259): 680-685.