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

Abstract

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

1. 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 an even interface between the two layers.
   2. After polymerization of the separating gel, prepare the stacking gel by pipetting 1.535 mL of ddH₂O, 625 mL of Tris-HCl (pH 6.8), 12.5 mL of 20% (w/v) SDS, 335 mL of 30% acrylamide/bisacrylamide (29:1) solution, 12.5 mL of 10% w/v ammonium persulfate, and 2.5 mL of TEMED. Remove the ethanol from the separating gels and add the stacking gel solution. Insert a comb with the desired number of pockets without introducing air bubbles. Allow polymerization for 20-30 min.
2. Load 4 µL of each sample and protein ladder into separate wells and run the gel(s) in Tris-Glycine running buffer (Table 1) at 144 V for 45 min at room temperature.
   NOTE: Stop the gel when the bromophenol band is about to migrate out of the gel.
3. Stain the gel(s) in a prewarmed Fairbanks solution A (Table 1) for 30 min on a rocker.
4. Decolor the gel(s) in a prewarmed Fairbanks solution D (Table 1) until the desired background (e.g., overnight) on a rocker.

Results

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chemicals/Reagents
Acetone	Fisher Scientific	67-64-1
30% Acrylamide/Bisacrylamide solution 29:1	Bio-Rad	1610156
Ammonium persulfate	Millipore Sigma	A3678-100G
Bluestain 2 Protein ladder, 5-245 kDa	GoldBio	P008-500
β-mercaptoethanol	Millipore Sigma	M6250-100ML
Bromophenol blue	GoldBio	B-092-25
Coomassie Brilliant Blue R-250	MP Biomedicals LLC	821616
Ethylenediamine tetra acetic acid (EDTA)	Sigma-Aldrich	SLBT9686
Glacial Acetic acid	Millipore Sigma	ARK2183-1L
Glycerol, 99%	Sigma-Aldrich	G5516-1L
Glycine	GoldBio	G-630-1
Isopropanol (2-Propanol)	Sigma	402893-2.5L
10x MOPS Buffer	Teknova	M2101
Sodium dodecyl sulfate (SDS)	Sigma-Aldrich	L3771-500G
Tetramethylethylenediamine (TEMED)	Millipore Sigma	T9281-50ML
Tris base	GoldBio	T-400-1
Material/Equipment
Power supply	ThermoFisher Scientific	EC105
Rocker	Alkali Scientific	RS7235
Small glass plate	Bio-Rad	1653311
Spacer plates (1 mm)	Bio-Rad	1653308
Spectrophotometer	Thermoscientific	3339053
Tabletop centrifuge for 15 mL centrifuge tubes	Beckman-Coulter
Vertical gel electrophoresis chamber	Bio-Rad	1658004
Vortexer	Fisher Vortex Genie 2	12-812
Thermomixer	Benchmark Scientific	H5000-HC
10 well comb	Bio-Rad	1653359