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

Published: April 30, 2023

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.

Representative 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
Fairbanks D (De-staining solution) 10% Glacial acetic acid solution
Lysis buffer 10 mM potassium phosphate (pH 6.5), 1 mM EDTA, 20% sucrose can be prepared and stored at room temperature for long term use. Add 1 mg/mL lysozyme and 50 u/mL Benzonase fresh before use. 
MOPS-g media 100 mL 10x MOPS, 10 mL 0.132 M K2HPO4, 10 mL 20% glucose, 0.5 mL 20 mM thiamine. Fill up to 1 L with ddH2O and sterile-filter
1x SDS sample buffer 6.5 mM Tris-HCl (pH 7), 10% glycerol, 2% SDS, 0.05% bromophenol blue and 2.5% β-mercaptoethanol. Stored at -20 °C.
12% SDS polyacrylamide gel preparation (for 2 gels) Separating gel: 5.1 mL ddH2O, 3.75 mL Tris-HCl (pH 8.8), 75 mL 20% w/v SDS, 6 mL 30% Acrylamide/Bisacrylamide solution 29:1 solution, 75 mL 10% w/v ammonium persulfate, 10 mL TEMED
Stacking gel: 1.535 mL ddH2O, 625 mL Tris-HCl (pH 6.8), 12.5 mL 20% w/v SDS, 335 mL 30% Acrylamide/Bisacrylamide solution 29:1 solution, 12.5 mL 10% w/v ammonium persulfate, 2.5 mL TEMED
SDS running buffer 25 mM Tris, 192 mM Glycine, 0.1% SDS in ddH2O. Store in room temperature.

Table 1: Buffer, Media, and Solutions. Recipes for buffer, media, and solutions used in this protocol.

Divulgaciones

The authors have nothing to disclose.

Materials

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

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Citar este artículo
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis for Protein Analysis: A Technique to Separate and Visualize Proteins Based on Molecular Weight. J. Vis. Exp. (Pending Publication), e21107, doi: (2023).

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