Overview
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 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.
- 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.
- 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. - Stain the gel(s) in a prewarmed Fairbanks solution A (Table 1) for 30 min on a rocker.
- Decolor the gel(s) in a prewarmed Fairbanks solution D (Table 1) until the desired background (e.g., overnight) on a rocker.
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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.
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Materials
| 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 |
