Western Blot: probing of the membrane
| Materials | |
|---|---|
| Staining Solution | 250 mL isopropanol; 100 mL acetic acid; 650 mL H2O; 0.03% Coomassie |
| Destaining Solution | 10% (v/v) isopropanol, 10% (v/v) acetic acid |
| Blocking Solution | TBS-T; 2% BSA; 0.05% NaN3 |
| TBS-Tween | 200 mL 10x TBS; 10 mL Tween; ad 2 L H2O |
| ECL-Solution | |
| H2O2 (30%) |
Procedure
- Upon completed transfer of the proteins to the PVDF membrane, the blotting apparatus is disassembled and the membrane is incubated for ~15 minutes at RT in staining solution with shaking
- Subsequently, the membrane is destained until the marker bands are visible and the background appears white
- The Protein Size Marker bands are marked on the membrane
- The membrane is quickly washed with TBS-T and then incubated in Blocking buffer (Blotto) for 1 h at RT or overnight at 4°C on a shaking platform
- The first antibody diluted in blocking buffer (1:500 – 1:5000) is added (usually 5 – 10 ml of antibody solution are sufficient) and the membrane is incubated overnight at 4°C on a shaking platform (cold room)
- The membrane is returned to RT, the solution containing the 1st antibody is saved in a 15 ml Falcon tube for reuse, and the membrane is washed three times for 10 – 20 min with ~50 ml TBS-Tween
- The membrane is incubated for 1 - 2 h with the HRP-conjugated secondary reagent (1:3,000 – 1:10,000 in 10 ml TBS-T) at RT on a shaking platform
- The membrane is washed again three times for 10 – 20 min each with ~50 ml TBS-T
- Add 10 ml of ECL-solution and 3 μL of H2O2 (30%) to the membrane and incubate about 2 min under shaking
- Place the ECL-soaked membrane on the ECL imager plate and record the luminescence. Transfer the image files in your directory on the central file server