There is an ongoing requirement in cell and molecular biology for the preparation of antibodies to use as probes for specific proteins. Two main strategies exist to raise appropriate antibodies. A complimentary deoxyribonucleic acid (cDNA), or gene sequence encoding the protein of interest can be expressed in a heterologous species,
From: Methods in Molecular Biology, vol. 295: Immunochemical Protocols, Third Edition. Edited by: R. Burns © Humana Press Inc., Totowa, NJ
usually bacteria, and the resultant purified protein used as an immunogen. Glutathione-S-transferase fusion proteins, for example, have been extensively used as immunogens. Alternatively, small synthetic peptides can be synthesized that contain amino acid sequences derived from the cDNA acid or gene. Such antipeptide antibodies crossreact with the corresponding intact native protein with surprisingly high frequency and have the additional advantage that the epitope recognized by the antibody is already well defined (1). In this way, antibodies can be raised against novel gene products that are specifically directed against sites of interest, for example, unique regions, highly conserved regions, active sites, extracellular domains, intracellular domains or regions of posttranslational modification, such as phosphorylation sites. Moreover, the ready availability of the peptide immunogen against which the antibody was raised means that sera can be rapidly and easily screened, for example, using an enzyme-linked immunosorbent assay for antipeptide activity. Free peptide can also be used to block antibody binding and so demonstrate immunological specificity, and it may be coupled to a solid support (e.g., agarose) to generate an affinity matrix for antibody purification. In this chapter, we describe the basic principles behind the design, synthesis, and use of synthetic peptides as immunogens and in this and the following chapter outline some of the basic methods used in our laboratories. These methods have been used for several years, with a considerable degree of success, by groups in our institute and elsewhere.
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