This unit details the use of bacterially produced fusion proteins for the production of antisera, allowing for the large-scale generation of affinity-purified antibodies to specific, targeted epitopes. The use of pET vectors containing a polyhistidine (His) or glutathione-S-transferase (GST) tag to construct bacterial expression plasmids are provided as prototypical examples of fusion protein methodology. The basic protocols provided in this unit describe: (1) transformation of E. coli for high-yield production of soluble fusion protein, (2) purification of soluble fusion proteins for use in immunization using chelated nickel or glutathione affinity chromatography (for His- and GST-tagged fusion proteins, respectively), (3) immunization of rabbits with purified fusion protein and collection of antisera, and (4) characterization of antisera for antibody specificity using immunoblotting techniques. Support protocols describe the purification of His-tagged insoluble fusion proteins for animal immunization and the construction and use of affinity columns for purifying antibodies using soluble fusion proteins.
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