BM Chemiluminescence Western Blotting Kit (Mouse/Rabbit) Protocol

Product No. 11520709001

Protocol

Preparation of membrane

Electrophoresis and electrotransfer

  • Carry out electrophoresis either use nondenaturing gels, SDS-PAGE or two-dimensional gels according to usual protocols.
  • Blot according to usual protocols.
  • After transfer blotting efficiency can be checked by reversibly staining the transferred proteins with Ponceau S solution.

General handling recommendations

Membrane handling requirements

  • Handle membrane only on the edges and with clean blunt-ended forceps
  • Clean scissors before cutting the membrane with an ethanol moistened towel
  • Wear powder free gloves
  • Make sure that always enough solution is present to cover the membrane entirely

Special handling of PVDF membrane

  • Wet hydrophobic PVDF membranes by a short rinse in methanol, whereby the membrane changes color from white to grey translucent. Then wet the membrane in transfer buffer for 3 min. Note: Do not use the membrane, if parts of the membrane remain white.
  • PVDF membranes must not dry out at any step. If drying occurs re-wet in 5% Tween 20 (v/v). This may, however, influence antibody binding.

Materials

     
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