14] Immunologic detection of specific proteins in cell extracts by fractionation in gels and transfer to paper

Abstract

This chapter describes the system of Reiser and Wardale, which allows efficient transfer of proteins from SDS-polyacrylamide gels to diazophenyl thioether (DPT) paper with only one hour of electrophoresis. This general approach is used for detecting specific proteins bound to diazobenzyloxymethyl (DBM) paper or sheets of nitrocellulose. When electrophoresis in the presence of SDS or urea is employed, the protein transfer technique is limited by the requirement that at least some antigenic determinants are either insensitive to denaturation and dissociation of subunits or are capable of refolding after transfer. Some proteins are difficult to transfer out of the gel at a particular pH, and others do not bind well to the solid support—for example, small proteins do not bind well to nitrocellulose. Proteins that interact with the affinity ligand are specifically transferred from the gel to the paper. This procedure is useful for analysis of both radio-labeled and unlabeled proteins.