Far-western blotting
Far-western blotting is a molecular biological method which is based on the technique of western blotting to detect protein-protein interaction in vitro. 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. Proteins in a cell lysate containing prey proteins are firstly separated by SDS or native PAGE, and transferred to a membrane, as in a standard WB. The proteins in the membrane are then denatured and renatured. The membrane is then blocked and probed, usually with purified bait protein(s). The bait proteins are detected on spots in the membrane where a prey protein is located, if the bait proteins and the prey protein together form a complex.
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).
Because cell extracts are usually completely denatured by boiling in detergent before gel electrophoresis, this approach is most useful for detecting interactions that do not require the native folded structure of the protein of interest.
External links
- Far-Western Blotting at the US National Library of Medicine Medical Subject Headings (MeSH)
- Overview at piercenet.com
- Overview at utoronto.ca
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