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 mRNAs. In Western blotting, 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 (Figure 6-12). The protein bands
are transferred to a nylon membrane by electrophoresis and
the individual protein bands are identified by flooding the
nitrocellulose membrane with radiolabeled or enzymelinked
polyclonal or monoclonal antibody specific for the
protein of interest. The Ag-Ab complexes that form on the
band containing the protein recognized by the antibody can
be visualized in a variety of ways. If the protein of interest 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 most
generally used detection procedures employ enzyme-linked
antibodies against the protein. After binding of the enzymeantibody
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.
The site of the protein of interest can be determined with
much higher sensitivity if a chemiluminescent compound
along with suitable enhancing agents is used to produce light
at the antigen site.