Far-western blot

The far-western blot, or far-western blotting, is a molecular biological method based on the technique of western blot to detect protein-protein interaction in vitro.[1][2] Whereas western blot uses an antibody probe to detect a protein of interest, far-western blot uses a non-antibody probe which can bind the protein of interest. Thus, whereas western blotting is used for the detection of certain proteins, far-western blotting is employed to detect protein/protein interactions.

Method

In conventional western blot, 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 blot employs non-antibody proteins to probe the protein 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).

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.

References
  1. Hall, Randy A. (2004). "Studying Protein–Protein Interactions via Blot Overlay or Far Western Blot". Protein-Protein Interactions. Methods in Molecular Biology. Vol. 261. pp. 167–174. CiteSeerX 10.1.1.380.553. doi:10.1385/1-59259-762-9:167. ISBN 978-1-59259-762-8. PMID 15064457.
  2. Machida, Kazuya; Mayer, Bruce J. (2009). "Detection of Protein-Protein Interactions by Far-Western Blotting". Protein Blotting and Detection. Methods in Molecular Biology. Vol. 536. pp. 313–329. doi:10.1007/978-1-59745-542-8_34. ISBN 978-1-934115-73-2. ISSN 1064-3745. PMID 19378071.


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