The far-western blot, or far-western blotting, is a molecular biological method based on the technique of

western blot The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detect ...

to detect protein-protein interaction ''in vitro''. Whereas western

blot Blot may refer to: Surname *Guillaume Blot (born 1985), French racing cyclist *Harold W. Blot (born 1938), served as United States Deputy Chief of Staff for Aviation *Jean-François Joseph Blot (1781–1857), French soldier and politician *Yvan ...

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 Gel electrophoresis is a method for separation and analysis of biomacromolecules ( DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF ...

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 An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...

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 ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...

'' using an

expression cloning Expression cloning is a technique in DNA cloning that uses expression vectors to generate a library of clones, with each clone expressing one protein. This ''expression library'' is then screened for the property of interest and clones of interest a ...

vector. The probe protein can then be visualized through the usual methods — it may be radiolabelled; it may bear a specific

affinity tag Protein tags are peptide sequences genetically grafted onto a recombinant protein. Tags are attached to proteins for various purposes. They can be added to either end of the target protein, so they are either C-terminus or N-terminus specific or ...

His His or HIS may refer to: Computing * Hightech Information System, a Hong Kong graphics card company * Honeywell Information Systems * Hybrid intelligent system * Microsoft Host Integration Server Education * Hangzhou International School, in ...

FLAG A flag is a piece of fabric (most often rectangular or quadrilateral) with a distinctive design and colours. It is used as a symbol, a signalling device, or for decoration. The term ''flag'' is also used to refer to the graphic design empl ...

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

, this approach is most useful for detecting interactions that do not require the native folded structure of the protein of interest.

References

External links

*
Overview at piercenet.com

Molecular biology techniques Protein methods {{molecular-biology-stub