Diagonal electrophoresis for the detection of protein disulfides

Abstract

Cysteines are one of the most rarely used amino acids in proteins, therefore when conserved in proteins they usually play critical roles in structure, function, or regulation of the protein. These cysteines or thiols can be reversibly oxidised to sulfenic acid (-SOH), thiyl radicals (-S) or nitrosothiols (-SNO) or form both inter-and intra-disulfide bridges (PSSP). The protein thiol groups PSSPs, represent a larger active redox pool than glutathione and are likely to be directly involved in cellular defence against oxidative stress. Diagonal electrophoresis is a relatively simple technique to analyze the formation of protein disulfides by sequential non-reducing/reducing electrophoresis. Proteins that do not form disulfides, electrophorese identically in both dimensions and form a diagonal after the second dimension, proteins that contained intra-chain disulfides lie above this diagonal, while those that formed inter-disulfides fall below the diagonal. This technique therefore allows for the detection and identification of protein disulfides.