- Use precast gels from BioRad (10 wells, 4-15 %)
- Warm the samples at 96ºC for 5 minutes
- Load samples in the wells (30 ul per well)
- Run with commercial running buffer 1x at 20 – 40mA
- Transfer to a nitrocelullose (or PVDF) membrane with Transblot Turbo (using the commercial buffer) –> high proteins or mixed proteins protocol
- Blotting: wash the membrane with water and cut it
- Blocking: blocking buffer Casein – TBS 1:1 1h
- Primary antibodies (diluted in Casein-TBS 1:1) –> 1h at RT or overnight at 4ºC
- Wash with TBS-tween0’1% (3×5′)
- Secondary antibody 1/7500 (diluted in Casein-TBS tween0’1%): 1h at RT
- Wash with TBS-tween0’1% 1x (2×5′)
- Wash with TBS 1x (1×5′)
- Read with Odyssey equipment
Handmade gels:
| 8 % separating gel | 4 % stacking gel |
| Tris pH 8.8 3M: 1.25ml Acrylamide 40%: 2 ml H2O: 6.65ml SDS 20%: 50ul PSA 10%: 100ul Temed: 10ul | Tris pH 6.8 2M: 0.75ml Acrylamide 40%: 1 ml H2O: 8.25 ml SDS 20%: 50ul PSA 10%: 100ul Temed: 10ul |
