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 Prepare a 12% SDS-polyacrylamide gel 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 ge…
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 </…
Declarações
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 |
Citar este artigo
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).