What is Polyacrylamide Gel Electrophoresis?
Polyacrylamide Gel Electrophoresis (PAGE) is a widely used technique for separating and analyzing proteins based on their size and charge. It involves the migration of proteins through a porous gel matrix made of cross-linked polyacrylamide under the influence of an electrical field.
The significance of PAGE lies in its ability to separate proteins with high resolution, allowing for the detection of minor differences in protein composition. It is a versatile and widely used technique that can be adapted for a variety of applications.
The applications of PAGE are diverse and include:
- Protein purification and characterization
- Identification of protein isoforms and variants
- Analysis of protein-protein interactions
- Determination of protein molecular weight
- Detection and quantification of protein contaminants in food and pharmaceuticals.
There are several variations of PAGE, including native PAGE, SDS-PAGE, and two-dimensional PAGE, each with different applications and requirements. This protocol will focus on SDS-PAGE.
Materials:
- Acrylamide/bis-acrylamide solution
- Tris buffer
- SDS (sodium dodecyl sulfate)
- TEMED (N,N,N',N'-tetramethylethylenediamine)
- APS (ammonium persulfate)
- Running buffer
- Protein sample
- Loading buffer
- Electrophoresis apparatus
- Power supply
- Staining solution
- Destaining solution
Procedure:
1. Prepare the separating gel by mixing appropriate volumes of acrylamide/bis-acrylamide solution, Tris buffer, SDS, TEMED, and APS according to the desired gel percentage and volume.
2. Pour the separating gel into the gel mold and insert the comb. Allow the gel to polymerize for approximately 30 minutes.
3. Prepare the stacking gel by mixing appropriate volumes of acrylamide/bis-acrylamide solution, Tris buffer, SDS, TEMED, and APS according to the desired gel percentage and volume.
4. Remove the comb from the separating gel and rinse the wells with running buffer.
5. Pour the stacking gel on top of the separating gel and insert the comb. Allow the gel to polymerize for approximately 30 minutes.
6. Remove the comb and rinse the wells with running buffer.
7. Prepare the protein sample by mixing it with loading buffer and heat the mixture at 95-100°C for 5-10 minutes to denature the proteins.
8. Load the protein sample into the wells using a micropipette.
9. Connect the electrophoresis apparatus to a power supply and fill the upper and lower chambers with running buffer.
10. Protein Gel and Imaging Analysis
11. Run the gel at a constant voltage according to the desired running time and voltage.
12. Once the gel run is complete, remove the gel from the apparatus and stain the proteins using a suitable staining solution.
13. Destain the gel in a suitable destaining solution to remove excess stain.
14. Visualize and analyze the separated proteins by imaging the gel and analyzing the bands.
Note: This protocol provides a general guideline for SDS-PAGE. Specific applications may require modification of certain parameters, such as gel percentage, running time, and staining method. It is important to refer to appropriate literature and consult with experienced colleagues when developing and optimizing a PAGE protocol for specific applications.
