Team:Freiburg/Protocols/Western Blot
Western Blot Towbin et al.
protocol for Western Blot Towbin et al.
remarks concerning the protocol (author, adapted from etc.)
material: chemicals, used kits, …
duration: pipapo…
- cut PVDF membrane and 4 whatman-papers in gel size
- PVDF membranes have to be activated in 100% MeOH before use
- Rinse membranes with blocking ubffer, Whatman-paper soaked in blocking buffer
- stack 2 whatman, membrane, gel and 2 whatman into the blotting apparatus
- (make sure there are no bubbles after adding each layer)
- blot for 1h (thin gels) (0.8mA/cm^2 Gel)
- block membrane ~ 30 min in blocking buffer
- 1st antibody incubation: 1h, RT or overnight 4°C
- wash membrane 3x 10 min in 1x TBS-T
- 2nd antibody incubation: 1h, RT or overnight 4°C
- wash membrane 3x 10min in 1y TBS-T
Staining with ECL:
- put membrane into prepared foil
- mix ECL-solution 1:1
- put mix on membrane; assure good distribution of the mix over the whole membrane
- incubation: 1min RT
- perform the read-out
Solutions: 10x Transfer buffer:
- 30.275g Tris base
- 144g Glycine
- bring up the volume to 1L with ddH2O
1xTransfer buffer:
- 700ml ddH2O
- 100ml 10x Transfer buffer
- 200ml Methanol
10x TBS
- 1M Tris/HCl pH 7.5
- 1.5M NaCl
Blocking Buffer:
- 5% dry milk powder in 1x TBS-T