protocol for Western blot towbin et al.
remarks concerning the protocol (author, adapted from etc.)

material: chemicals, used kits, …
duration: … min

• 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