Western Blotting
- Identification of a specific protein in a complex mixture of proteins can be accomplished by a technique known as Western blotting, named for its similarity to Southern blotting, which detects DNA fragments, and Northern blotting which detects mRNA.
- In Western, a protein mixture is electrophoretically separated on an SDS-polyacrylamide gel (SDS-PAGE), a slab gel infused with sodium dodecyl sulfate (SDS), a dissociating agent.
- The protein bands are transferred to a nitrocellulose membrane by electrophoresis, and the individual protein bands identified by flooding the membrane with radiolabeled or enzyme-protein of interest.
- The Ag-Ab complexes that form on the band containing the protein recognized by the antibody can was bound by a radioactive antibody, its position on the blot can be determined by exposing the membrane to a sheet of x-ray film, a procedure called autoradiography.
- However, the linked antibodies against the protein.
- After binding of the enzyme antibody conjugate, addition of a chromogenic substrate that produces a highly colored and insoluble product causes the appearance of a colored band at the site of the target antigen.
- Even greater sensitivity can be achieved if a chemiluminescent compound with suitable enhancing agents is used to produce light at the antigen site.
- Western blotting can also identify a specific antibody in a mixture.
- In this case, known antigens of well defined molecular weight are separated by SDS-PAGE and blotted onto nitrocellulose.
- The separated bands of known antigens are then probed with the sample suspected of containing antibody specific for one or more of these antigens.
- Reaction of an antibody with a band is detected by using either radiolabeled or enzyme-linked secondary antibody that is specific for the spiciest of the antibodies in the test sample.
- The most widely used application of this procedure is in confirmatory testing for HIV, where western blotting is used to determine whether the patient has antibodies that react with one or more viral proteins.
