Biotechnology and Bioinformatics (1+1)

4.2.12.3. Western blot ting

Western blotting is a technique by which protein s can be transferred from a polyacrylamide gel to a sheet of nitrocellulose so that a replica of the original gel pattern is obtained. A wide variety of analytical procedures can then be applied to immobilized protein. In this technique, a sheet of nitro-cellulose is placed against the surface of a SDS-PAGE protein fractionation gel and a current applied across the gel (at right angles to its face). This causes the proteins to move out of the gel and intro the nitrocellulose, where they bind firmly by non-covalent forces.

The technique involves three steps:

• protein separation by SDS-PAGE,
• blotting and
• immunoassay.
• To detect a specific protein, an antibody to that protein must be available. An antibody can either be produced for the protein of interest or some times purchased commercially. The nitrocellulose membrane itself has many non-specific sites that can bind proteins, including antibodies. These sites must be blocked with a non-specific protein solution such as re-hydrated milk. The primary antibody is added in the milk solution and binds to the protein of interest. The antibody protein complex is detected using a secondary antibody that has a label attached to it. Often a reporter enzyme such as alkaline phosphatase is linked to the secondary antibody, and the addition of lumiphos or X-phos to the blot allows detection of the protein band.

Application

i) screening of hybridoma clones .

ii) used as a diagnostic tool for various pathological conditions.

iii) ease of processing for autoradiography .

iv) include hormone -receptor, cyclic AMP-receptor and protein-nucleic acid interactions can be analysed.

v) used to distinguish species of piscine trypanosomes.