Far-western blotting

A Far Western blot is a biochemical method of detecting protein-protein interactions.

Principle

A sample is separated by SDS -PAGE or 2D - gel electrophoresis and transferred by Western blot to a membrane. The blot is incubated after blocking with a purified protein. This protein for the detection of a specific antibody for immunostaining is required, or the protein has previously been labeled with a radionuclide, by biotinylation, or with a fluorophore. By detection of the protein which has bound to its interacting partners on the blot, are obtained by comparison with the size marker, the molecular weights of the binding partners, and by mass spectrometry or by Edman degradation and part of an identifiable sequence.

As a result of denaturation in SDS -PAGE and subsequent, often incomplete renaturation some protein folds are not restored, this method for denaturing insensitive interactions such as in linear or phosphorylated sequences is suitable. Alternatively may be carried out for the separation of the proteins to obtain the native protein structure, a native gel electrophoresis, however, a size determination on the size marker is not possible with it.