Far-western blotting
Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]
Far-western blotting is a molecular biological method which is based on the technique of Western blotting. While usual western blotting uses an antibody to detect a protein of interest, far-western blotting uses a non-antibody protein, which can bind the protein of interest. Thus, whereas western blotting is used for the detection of certain proteins, far-western blotting is rather employed to detect protein:protein interactions.
Method
In conventional western blotting, gel electrophoresis is used to separate proteins from a sample; these proteins are then transferred to a membrane in a 'blotting' step. In a western blot, specific proteins are then identified using an antibody probe.
Far-western blotting employs non-antibody proteins to probe the protein(s) of interest on the blot. In this way, binding partners of the probe (or the blotted) protein may be identified. The probe protein is often produced in E coli using an expression cloning vector.
The probe protein can then be visualized through the usual methods— it may be radiolabelled; it may bear a specific affinity tag like His or FLAG for which antibodies exist; or there may be a protein specific antibody (to the probe protein).
External links
- Far-Western+Blotting at the US National Library of Medicine Medical Subject Headings (MeSH)
- Overview at piercenet.com
- Overview at utoronto.ca