SDS-PAGE Protocol

The basic protocol for Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) is commonly used to separate proteins according to their molecular weight.

Materials Needed:

• Acrylamide solution (desired concentration)
• SDS buffer (Tris-glycine running buffer, 10X)
• 10% SDS solution
• 10% ammonium persulfate solution (APS)
• TEMED
• Protein samples
• 2x Laemmli sample buffer (4% SDS, 20% Glycerol, 10% 2-Mercaptoethanol, 0.004% bromophenol blue and 0.125M Tris HCl)
• Molecular Weight Standard
• Staining solution (Coomassie blue or silver stain)

Steps:

1. Preparing the Resolving Gel:
   a. Mix the resolving gel solution that includes Acrylamide, Tris-Cl (pH 8.8), 10% SDS, and deionized water.
   b. Degas the mixture for ~10 minutes.
   c. Add TEMED and 10% ammonium persulfate (APS) to initiate polymerization and mix well.
   d. Pour the resolving gel mixture into the assembly, insert a comb to create wells, and let it polymerize.
2. Preparing the Stacking Gel:
   a. Once the resolving gel has polymerized, mix the stacking gel solution that includes Acrylamide, Tris-Cl (pH 6.8), 10% SDS, and deionized water.
   b. Degas the mixture for ~10 minutes.
   c. Mix well with TEMED and 10% APS to initiate polymerization.
   d. Pour the stacking gel mixture on top of the resolved gel.
   e. Insert the comb and let it polymerize.
3. Preparing the Samples:
   a. Boil the protein samples with 2x Laemmli sample buffer for around 5 minutes.
   b. Cool down the samples on ice.
4. Gel Electrophoresis:
   a. Remove the comb and rinse the wells with running buffer.
   b. Load samples into the wells in a gel along with the molecular weight standard.
   c. Run the gel at constant current, starting at a lower voltage (80-120V) until the samples have entered the resolving gel, then increase to a higher voltage (150-200V).
5. Staining:
   a. Staining the gel with protein-dye like Coomassie blue or a silver stain to visualize the separated protein bands.

This is a generalized protocol; parameters can differ based on the nature and type of protein samples, and acrylamide concentration could influence on the separation of proteins. Please make sure to use the appropriate safety protocols when handling chemical reagents. Always refer to specific guidelines or protocols from your lab or product manufacturer.

Read More: SDS-PAGE Gel Preparation