Protein Protocols Protein Protocols

Reutilization of Western Blots 439
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Reutilization of Western Blots After Chemiluminescent
Detection or Autoradiography
Scott H. Kaufmann
1. Introduction
Western blotting (also called immunoblotting) is a widely utilized laboratory procedure
that involves formation and detection of antibody–antigen complexes between
antibodies that are initially in solution and antigens that are immobilized on derivatized
paper (reviewed in refs. 1–3). This procedure is most commonly performed by
sequentially subjecting a complex mixture of polypeptides to three manipulations:
(1) electrophoretic separation, usually through polyacrylamide gels in the presence of
sodium dodecyl sulfate (SDS); (2) electrophoretic transfer of the separated polypeptides
to thin sheets of nitrocellulose or polyvinylidene fluoride (PVDF); and (3) reaction
of the sheets sequentially with one or more antibody-containing solutions. Because
these are laborious, low-throughput procedures, there has been considerable interest
over the past 15 yr in increasing the information gained from immunoblots in various
ways. The approaches to this problem have included removal of bound antibodies so
that immobilized polypeptides can be reprobed with additional antisera (last reviewed
in ref. 4), the sequential (5) or simultaneous (6) use of multiple antisera to detect
different antigens, and the development of higher density filter manifolds to permit
more spots to be placed in the same area when purified proteins are applied to
filters by spot adsorption. After a brief review of critical parameters for successful
immunoblotting, some of these approaches are briefly discussed and illustrated.
Because of its versatility, immunoblotting is widely employed in biological studies.
Critical to the success of this procedure is the quality of the immunological reagents
utilized. With a high quality antiserum or monoclonal antibody, this approach can be
utilized to determine whether an antigen of interest is present in a particular biological
sample, to monitor the purification of the antigen, or to assess the location of epitopes
within the antigen after chemical or enzymatic degradation. With suitable immunological
probes, this same approach can be utilized to search for proteins that bear
a particular physiological or pathological posttranslational modification, for example,
phosphorylation (7) or nitrosylation of tyrosine (8), covalently attached glycosylphosphatidylinositol
(9), or modification by D-penicillamine metabolites (10). Conversely,
immunoblotting can also be utilized to determine whether antibodies that recognize a
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
439