The past few years have seen a substantial increase in the commercial availability of antibodies to a wide variety of antigens. In addition, the increasing use of epitope tags as a means to identify proteins in gene expression studies sometimes bypasses the need for de novo generation of antibodies against a given antigenic target. Nevertheless, the production of specific antibodies to a protein of interest or to particular regions of a protein of interest remains an important tech-

From: Methods in Molecular Biology, vol. 295: Immunochemical Protocols, Third Edition. Edited by: R. Burns © Humana Press Inc., Totowa, NJ

nique for the generation of specific tools to aid the characterization of biological processes at the cellular and molecular level.

Immunization protocols often vary substantially between laboratories, and different protocols can often produce satisfactory results. Although there are few hard and fast rules, the methods described here are designed to give optimal results with minimal discomfort to the donor animal and they have been used successfully in our institute and elsewhere for a number of years (1-5). Despite the fact that the immunization of, for example, goats or sheep produces a greater volume of antiserum, polyclonal antisera raised in rabbits offers the advantage of the ready availability and general high quality of a wide variety of antirabbit secondary detection reagents. This can be a significant issue, especially if the antibodies are to be used for purposes more demanding than conventional Western blotting or immunoprecipitation.

Short peptides are generally poor immunogens and must be conjugated to carrier proteins, which provide the necessary helper T-cell epitopes for an efficient antibody response. Keyhole limpet hemocyanin (KLH) is a very common and effective carrier protein to use for this purpose, although other proteins, such as bovine serum albumin and thyroglobulin also work well. Purified proteins, for example, those isolated by means of bacterial expression, normally don't require coupling to a carrier before immunization. Nevertheless, conjugation may be worth considering should the protein be rather short or if it is thought to be only weakly immunogenic.

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