SDS-PAGE ANALYSIS
PRINCIPLE
SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a biochemical assay to analysis the purity and apparent molecular weight of protein. In this assay, sodium dodecyl sulfate (SDS) is necessary that is an anionic detergent and is used to linearize the proteins and impart a negative charge. The relative migration distance of a protein is negatively proportional to its molecular weight. In order to visually estimate molecular weight, protein maker and coomassie brilliant blue are used in SDS-PAGE. Coomassie brilliant blue is an anionic dye, which binds to proteins and make them visualized as blue bands on a clear background.
MATERIALS
Reagents
- 30% Acrylamide
- 1.5 M Tris-HCl, pH 8.8
- M Tris-HCl, pH 6.8
- 10% SDS
- 10% Ammonium Persulfate
- TEMED
- Protein Marker
- Loading Buffer
- Running Buffer
- Coomassie Stain Solution
- Destain Solution
Instruments and Consumables
- Electrophoresis Chamber
- Eppendoff
- Oscillating Table
- Incubation Plate
METHODS
Casting the gel
1. Assemble glass plates and spacers in gel casting apparatus.
2. Mix the components for the resolving gel as described above.
3. Pour the resolving gel mixture into the gel plates to a level 2 cm below the top of the shorter plate.
4. Pace a layer of ddH2O over the top of the resolving gel.
5. Allow resolving gel to stand 30 min at room temperature.
6. Drain the ddH2O from top of the resolving gel. Rinse with ddH2O, drain, and wick any remaining ddH2O away with absorbent paper.
7. Mix components for stacking gel.
8. Pour stacking gel solution into top of running gel. Insert comb to the top of the spacers.
9. Allow gel to stand for at least 1 hour at room temperature.
Preparing samples
10. Place a volume of protein solution into a tube.
11. Add a volume of loading buffer.
12. Incubate tubes in boiling water for 10 min.
13. Centrifuge at 12,000g for 30s.
Running the gel
14. Remove comb and assemble cast gel into Electrophoresis chamber.
15. Add freshly prepared running buffer to both chambers of the apparatus.
16. Load the prepared samples into the wells of the gel.
17. Run the gel at 90V until the dye front migrates into the running gel, and increase to 150V until the dye front reaches the bottom of the gel.
Staining and destaining the gel
18. Remove the run gel from the apparatus and remove the spacers and glass plates. Place the gel into an incubation plate.
19. Add staining solution to completely submerge the gel.
20. Stain for 15 min with gentle shaking.
21. Pour off and save the stain.
22. Add destain solution to completely submerge the gel.
23. Destain for 10 min with gentle shaking. Pour off and discard the destain solution.
24. Repeat step 23 until the gel is visibly destained.
25. Pour off and discard the destain solution. Rinse with ddH2O.
26. Record the results.
