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.
1. Separation and visualization of extracted protein aggregates using SDS-PAGE
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.
The authors have nothing to disclose.
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 |