Far-western blotting
Encyclopedia
Far-Western blotting is a molecular biological method which is based on the technique of Western blot
ting. 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.
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).
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.
Western blot
The western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...
ting. 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 electrophoresisGel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
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
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
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
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
using an expression cloning
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 recovered for further analysis...
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
Polyhistidine-tag
A polyhistidine-tag is an amino acid motif in proteins that consists of at least five histidine residues, often at the N- or C-terminus of the protein. It is also known as hexa histidine-tag, 6xHis-tag, and by the trademarked name His-tag...
or FLAG
FLAG-tag
FLAG-tag, or FLAG octapeptide, is a polypeptide protein tag that can be added to a protein using recombinant DNA technology. It can be used for affinity chromatography, then used to separate recombinant, overexpressed protein from wild-type protein expressed by the host organism...
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
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
, this approach is most useful for detecting interactions that do not require the native folded structure of the protein of interest.