05 Nov

Western Blot

Western blot involves the transfer to PVDF membrane or Nitrocellulose membrane from a gel after the proteins of interest have been separated by SDS PAGE.  After transfer, the membrane is probed with antibodies for detection of a specific protein.  PVDF and Nitrocellulose are used as blotting membrane because they have non specific binding of proteins, they bind all proteins equally well.  PVDF would be preferred when the proteins of interest are hydrophobic.  The Western blot procedure involves placing the transfer membrane against the gel and sandwiching the gel and membrane between sponges.  Transfer can be made by capillary action of the transfer buffer through the gel and onto the membrane but this is slow.  A better method is to use electroblotting where an electric current is used to pull the proteins out of the gel and on to the blotting paper.  The blotting paper must then be stained to insure that the proteins have actually transferred and then de-stained.  The membrane must be blocked with protein such as BSA or milk protein to insure that the antibody probe is not bound by the membrane and the only binding is by the target protein.  The primary antibody (host could be mouse or goat. . .) to the protein of interest is then introduced.  A secondary antibody (anti mouse or goat . . .) conjugated to an enzyme such as horseradish peroxidase or alkaline phosphatase is added.  A chemiluminescent substrate is added for visualization and the membrane is photographed.

Robin Prymula