Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis for Protein Analysis: A Technique to Separate and Visualize Proteins Based on Molecular Weight

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Samenvatting

Source: Sultana, S. et al. Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor. J. Vis. Exp. (2021).

This video demonstrates a denaturing polyacrylamide-based separation technique for proteins. This technique helps in the preliminary identification of proteins based on their molecular weights.

Protocol

1. Separation and visualization of extracted protein aggregates using SDS-PAGE

Prepare a 12% SDS-polyacrylamide gel
1. For two separating gels, pipette 5.1 mL of double-distilled water (ddH₂O), 3.75 mL of Tris-HCl (pH 8.8), 7.5 mL of 20% (w/v) SDS, 6 mL of 30% acrylamide/bisacrylamide (29:1) solution, 75 mL of 10% w/v ammonium persulfate, and 10 mL of tetramethylethylenediamine (TEMED) into a 15 mL centrifuge tube and mix gently without introducing air bubbles. Pour the gel using a 1 mL pipet within the glass plates, leaving the upper 2 cm free of the mixture. Add 70% ethanol on the top of the separating gel and allow a....

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Resultaten

Solutions	Recipes
Buffer A	10 mM potassium phosphate (pH 6.5), 1 mM EDTA
Buffer B	Buffer A containing 2% Nonidet P-40. Can be stored in room temperature for later use.
Fairbanks A (Staining solution)	25% isopropanol, 10% Glacial Acetic acid, 1.4 g Coomassie R-250