Methods to identify and avoid artifactual formation of interchain disulfide bonds when analyzing proteins by SDS-PAGE.

Amicyanin is a monomeric protein of known structure which possesses a single cysteine that serves as a ligand to copper in its native state. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of amicyanin after denaturation in the presence and absence of beta-mercaptoethanol, however, indicated that this protein was a dimer which was covalently linked by interchain disulfide bonds. This artifact was caused by exposure during denaturation of the free cysteine that normally binds copper and subsequent formation of a disulfide bond between otherwise unliked monomers. This phenomenon is documented, and additional control experiments are proposed to identify and avoid this artifact when using SDS-PAGE to analyze the subunit structure of proteins.