In the late 1970s - beginning of the 1980s, we’d developed a pioneer approach to prepare antibodies against monooxygenase molecular isoforms without laborious purification of CYPs. To achieve the point, we’d conducted immunization with protein band from SDS-PAGE (bands were preliminary characterized by isozyme-specific peptide maps). Thereafter in the 1990s-2000s, I’ve been deeply involved in preparation of the recombinant CYPs, and therefore lost a little bit the track on current procedure with SDS-PAGE-CYPs. Namely, if one need to inject mice with a protein band excised from SDS-PAGE gel, is it necessary to use Freund's adjuvant while using this protocol, won't the acrylamide itself act as an immunogen (can it serve as a mild adjuvant and/or booster after 6 weeks)? Some people stain gel, fix with 2% glutaraldehyde and destain, then cut the band of interest, pulverize in an Eppendorf tube, freeze-dry (lyophilize), mix with PBS and adjuvant, emulsify and inject. Others rather fix first, wash to remove glutaraldehyde, stain with reversed staining (e.g. zinc, CuCl2) and then cut. And how much protein might be cumulated in several parallel bands run, in terms of would it be enough to inoculate depending whether one is injecting rabbits or mice.
polyacrylamide gel electrophoresis can be difficult to interpret when looking at isoenzyme specific peptide maps.
Glutaraldehyde should be removed .
Use rabbits rather than mice .
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