SDS-PAGE Protocol
Principle
SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is a biochemical assay to analyze 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. 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 makes them visualized as blue bands on a clear background.
Materials
Reagents
- 30% Acrylamide
- 1.5 M Tris-HCl, pH 8.8
- 1 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 (glass plates, casting stand, comb)
- Centrifuge
- Water Bath
- Oscillating Table
- Incubation Plate
- Power Supply
- Filter Paper
- Microcentrifuge Tubes
- Gel Documentation Camera System
- White Light Transilluminator
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 the resolving gel to stand for 30 min at room temperature.
6. Drain the ddH2O from the 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 the stacking gel solution into the top of the running gel. Insert the comb to the top of the spacers.
9. Allow the 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 the comb and assemble the 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 running gel from the apparatus and remove the spacers and glass plates. Place the gel into an incubation plate.
19. Image the gel on a gel documentation camera system or protein staining.
20. Add staining solution to completely submerge the gel.
21. Stain for 15 min with gentle shaking.
22. Pour off and save the stain.
23. Add destain solution to completely submerge the gel.
24. Destain for 10 min with gentle shaking. Pour off and discard the destain solution.
25. Repeat step 24 until the gel is visibly destained.
26. Pour off and discard the destain solution. Rinse with ddH2O.
27. Record the results, illuminate and photograph the gel using a white light transilluminator. 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 the resolving gel to stand for 30 min at room temperature.
6. Drain the ddH2O from the 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 the stacking gel solution into the top of the running gel. Insert the comb to the top of the spacers.
9. Allow the 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 the comb and assemble the 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 running gel from the apparatus and remove the spacers and glass plates. Place the gel into an incubation plate.
19. Image the gel on a gel documentation camera system or protein staining.
20. Add staining solution to completely submerge the gel.
21. Stain for 15 min with gentle shaking.
22. Pour off and save the stain.
23. Add destain solution to completely submerge the gel.
24. Destain for 10 min with gentle shaking. Pour off and discard the destain solution.
25. Repeat step 24 until the gel is visibly destained.
26. Pour off and discard the destain solution. Rinse with ddH2O.
27. Record the results, illuminate and photograph the gel using a white light transilluminator.
Application
- Protein Separation
Proteins can be separated using SDS-PAGE based on their size. Smaller proteins move more quickly through the gel matrix, while larger proteins move more slowly. As a result, proteins separate into distinct bands according to their molecular weights.
- Protein Purity and Composition Analysis
By running both the protein sample and a molecular weight marker (containing proteins of known sizes) simultaneously, researchers can verify the size and integrity of their target protein and identify any contaminating proteins. Additionally, SDS-PAGE can determine the stoichiometry of protein complexes or verify the presence of specific subunits within a protein complex.
- Protein Quantification
SDS-PAGE can be utilized for semi-quantitative analysis of protein samples. By comparing the intensity of protein bands against standard curves generated from known amounts of proteins, researchers can estimate the relative abundance of target proteins within a sample.
- Protein Western Blotting
After electrophoresis, proteins separated on the SDS-PAGE gel can be transferred (blotted) onto a membrane, a technique known as western blotting or immunoblotting. Western blotting is widely employed for protein identification, protein-protein interaction studies, and the analysis of post-translational modifications.
- Protein Characterization
SDS-PAGE is often used as an initial step in protein characterization, as it provides important information about the size and purity of protein samples. The separated protein bands can be excised from the gel and subjected to downstream analyses such as mass spectrometry for protein identification or proteomic studies.
- Medical Diagnostics
HIV proteins are separated by SDS-PAGE and subsequently detected by Western Blot using the patient's HIV-specific antibodies in the HIV test. Proteinuria SDS-PAGE determines the amounts of various serum proteins in the urine.
Case Study
Fig. 1 SDS-PAGE used for Protein Purity analysis. 1
Fig. 2 GSK3β and β-catenin immunoprecipitation experiment. 2
References
- Mei, Guang-Ming et al. "Structural Characterization and In Vitro Antioxidant Activity of Metallothionein from Oratosquilla oratoria." Molecules (Basel, Switzerland) vol. 27,7 2320. 3 Apr. 2022. Distributed under Open Access license CC BY 4.0, without modification.
- Junjie, Li et al. "Citrus alkaline extracts improve LPS-induced pulmonary fibrosis via epithelial mesenchymal transition signals." Chinese medicine vol. 18,1 62. 29 May. 2023. Distributed under Open Access license CC BY 4.0, with modification. The image was modified by extracting and using only Part F of the original image.
