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Protocol: Silver Staining
Gel making
Gel Ingredients (choose percentage according to the size of the protein)
| 4-40 kDA | 20% |
| 12-45 kDA | 15% |
| 10-70 kDA | 12.5% |
| 15-100 kDA | 10% |
| 25-200 kDA | 8% |
| Separating gel | |
| Gel percentage | 7.5 % |
| 30% Polyacrylamide | 10 mL |
| 1.5M Tris (pH 8.8) | 10 mL |
| 10% Ammonium persulfate | 0.4 mL |
| 10% SDS | 0.4 mL |
| TEMED | 0.038 mL |
| H2O | 19.2 mL |
| Total volume | 40 mL |
| Stacking gel | |
| Gel percentage | 5 % |
| 30% Polyacrylamide | 1.36 mL |
| 1M Tris (pH 6.8) | 1 mL |
| 10% Ammonium persulfate | 0.08 mL |
| 10% SDS | 0.08 mL |
| TEMED | 0.008 mL |
| H2O | 5.44 mL |
| Total volume | 8 mL |
- Loading Plan
- Lane 1: Ladder
- Lane 2: Empty well
- Lane 3: 10microL of VP16 only
- Lane 4: 25microL of VP16 only
- Lane 5: Nontransfected CHO cell - High concentration (5million)
- Lane 6: Sample 1 48h of expression
- Lane 7: Sample 2 48h of expression
- Lane 8: Sample 1 72h of expression
- Lane 9: Sample 2 72h of expression
- Lane 10: Nontransfected CHO cell - Same concentration as samples
Result: The lane 9 (Lovtap-VP16 transfected CHO cell) and lane 10 (non-transfected CHO cell) have intensity difference around 30~35kDa area.
