SDS-PAGE Gel Preparation
1. Prepare the separation gel (10%). Mix in the following order:
| H2O | 4.1 mL |
| Acrylamide/bis (30% 37.5:1; Bio-Rad) | 3.3 mL |
| Tris-HCl (1.5 M, pH 8.8) | 2.5 mL |
| SDS, 10% | 100 µL |
| N,N,N′,N′-tetramethylethylene-diamine (TEMED) (Bio-Rad) | 10 µL |
| Ammonium persulfate (APS), 10% | 32 µL |
After adding TEMED and APS to the SDS-PAGE separation gel solution, the gel will polymerize quickly, so add these two reagents when ready to pour.
2. Pour gel, leaving ∼2 cm below the bottom of the comb for the stacking gel. Make sure to remove the bubbles.
3. Layer the top of the gel with isopropanol. This will help remove bubbles at the top of the gel and keep the polymerized gel from drying out.
In about 30 minutes, the gel should be completely polymerized.
4. Remove the isopropanol and wash out the remaining traces of isopropanol with distilled water.
5. Prepare the stacking gel (4%). Mix in the following order:
| H2O | 6.1 mL |
| Acrylamide/bis (30%, 37.5:1) | 1.3 mL |
| Tris–HCl (0.5 M, pH 6.8) | 2.5 mL |
| SDS, 10% | 100 µL |
| TEMED | 10 µL |
| Ammonium persulfate (APS), 10% | 100 µL |
6. Pour stacking gel on top of the separation gel.
7. Add combs to make wells. In ∼30 to 30 minutes, the stacking gel should become completely polymerized.
8. Clamp gel into the apparatus and fill both buffer chambers with gel-running buffer according to the instructions for the specific apparatus.
9. Load samples and molecular mass protein markers into wells for separation by electrophoresis.
Read More: SDS-Gel Electrophoresis Protocol
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