Page 1409 - Veterinary Immunology, 10th Edition

transcriptase PCR analysis. Its use is discussed in Chapter 33.
VetBooks.ir Western Blotting

One solution to the problem of identifying protein antigens in a
complex mixture is by use of a technique called Western blotting.

This is a three-stage primary binding test (Fig. 42.10). Stage 1
involves electrophoresis of a protein mixture on gels so that each
component is resolved into a single band. Stage 2 involves blotting
or transfer of these protein bands to an immobilizing nitrocellulose
membrane. This is accomplished by placing the membrane on top

of the gel and sandwiching the two between sponges saturated
with buffer. The membrane-gel sandwich is supported between
rigid plastic sheets and placed in a buffer reservoir, and an electrical

current is passed between the sponges. The protein bands are
transferred from the gel to the membrane without loss of resolution.

FIG. 42.10 The Western blotting technique. Serum is separated by
electrophoresis and blotted onto nitrocellulose paper; the antigen
bands are revealed by use of specific antibody and an enzyme- or
isotope-labeled antiglobulin. The blotting stage may be a passive
transfer or an electric potential may be used to accelerate the
blotting process.

The third stage involves visualization of transferred antigens by
means of an enzyme immunoassay or radioimmunoassay. When an
enzyme immunoassay is employed, the membrane is first incubated

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