Diagonal Polyacrylamide-Dodecyl Sulfate Gel Electrophoresis for the Identification of Ribosomal Proteins Crosslinked with Methyl-4-Mercaptobutyrimidate

Abstract

A diagonal polyacrylamide-dodecyl sulfate gel electrophoresis procedure is described. Its utility is related to the use of the reagent methyl-4-mercaptobutyrimidate as a protein-protein crosslinking reagent. Crosslinking with this reagent occurs through the formation of intermolecular disulfide bonds. Oxidized proteins are separated in one dimension by electrophoresis under non-reducing conditions and in the second dimension under reducing conditions. All proteins except those derived from crosslinked species fall on a diagonal. Methods are described for the identification of the separated monomeric components of crosslinked species. The technique has been applied to the 30S ribosomal subunit of Escherichia coli, and the new crosslinked dimer, S4-S13, has been identified.