SDS PAGE ELECTROPHORESIS OF PROTEIN

Описание

SDS PAGE - electrophoresis is an analytical method for isolation and separation of proteins in biological sample. This method frequently used in life science researchers, scientist, microbiologist, forensic science etc. In this video i am presenting about principle, instrumentation, application and factors affecting electrophoresis with reference to SDS PAGE.

Protocol

Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis [SDS-PAGE]
SDS-PAGE was performed by the method of Laemmli (1970).

Reagents

Preparation of stock solution and buffers

1. 30 % Monomer
a. Acrylamide: 29.2 g
b. N, N’-methylene-bis-acrylamide: 0.8 g dissolved in water, made upto 100 ml and filtered with Whatman No.1 filter paper.

2. Separating gel buffer
a. Tris-HCl: 1.5 M, pH 8.8
18.171g of Tris was taken and dissolved in 60 ml of H2O and adjusted the pH to 8.8 with HCl and made up to 100 ml with H2O

3. Stacking gel buffer
a. Tris-HCl: 1 M, pH 6.8
6.57g of Tris was dissolved in 60 ml of H2O, adjusted the pH to 6.8 with HCl and made up to 100 ml with H2O.

4. 10 % SDS solution: 1g of SDS in 10 ml of distilled water.

5. N,N,N'N'-Tetra methylethylene diamine (TEMED)

6. 10 % Ammonium per sulfate: 1g of APS in 10 ml of distilled water.

7. Electrophoresis buffer, pH 8.3 Tank Buffer
a. Tris: 3.28 g
b. Glycine: 14.41 g
c. SDS: 1 g
Dissolved in minimum amount of water (500 ml) and then added SDS. Allowed to settle and dissolve and made up to 1 litre.

8. Sample Buffer 2X - 10 ml (SSB)
a. Tris (1M, pH 6.8): 3.1 ml
b. 10 % SDS: 4 ml
c. Glycerol (20 %): 2.0 ml
d. β-Mercaptoethanol: 0.5 ml
e. Bromophenol blue: 2.0 mg
f. Distilled water: 0.4 ml

9. Staining solution (100 ml)
a. Coomassie Brilliant Blue R-250: 250 mg
b. Methanol: 50 %
c. Acetic acid: 10 %
d. Distilled water: 40 %

10. Destaining solution (100 ml)
a. Methanol: 50 %
b. Acetic acid: 10 %
c. Distilled water: 40 %


Procedure
Gel plates of dimension 17 X 17cm with 1mm spacers were set up to cast the gel. 12 % resolving gel solution was prepared and poured in between glass plates (sandwich) in gel apparatus. The gel was allowed to polymerize for 20 min. The polymerized resolving gel was overlaid with 5 % stacking gel. The comb was inserted without any air bubble in between the plates containing gel solution. The stacking gel was allowed to polymerize. The comb was fixed properly to get clear-cut wells and the stacking gel allowed polymerizing for 20 min. After polymerization, the comb was removed and the wells formed were used for loading the sample.

Sample preparation and loading
The protein sample was mixed with equal volume of buffer, boiled for 5 min, cooled and loaded. (The final concentration of the sample buffer in the prepared sample should be 1X).

Electrophoresis

A constant voltage of 50 V and 10 A current was supplied by means of a power pack. The anode and cathode buffer tank were filled approximately with running reservoir buffer and checked regularly for the migration of bromophenolblue dye towards anode. The current and the respective voltage may be switched to 20 A and 100 V after the sample entered the resolving gel and the run was stopped when the tracking dye reached the bottom of the gel.
After confirming that the stacking gel had set properly, the comb was removed slowly and the set up was placed in an electrophoretic apparatus.
Coomassie brilliant blue staining of gel
0.25 % CBB-R250 in methanol, acetic acid and water in the ratio of 5:1:4 (V/V/V) respectively were prepared. The gel was soaked in the staining solution for about 4 hr. The gel containing the staining solution was the poured off completely and the gel was soaked in destaining solution (methanol, acetic acid and water) 5:1:4 (V/V/V). The gel was kept on a rotary shaker until protein bands were visualized clearly. The gel was then fixed in 7.5 % acetic acid and stored at room temperature.
Confirming the protein band separation and the band at the molecular weight of protein of interest, the SDS - PAGE was run as indicated with the same sample for the western blotting.