Team:Freiburg/Protocols/Western Blot

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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
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