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.
Kumar
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MA
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505.
Davidson
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VL
D016477:Artifacts; D001426:Bacterial Proteins / Q000032:analysis; D003545:Cysteine / Q000378:metabolism; D003553:Cystine / Q000138:chemical synthesis; D004220:Disulfides / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D011489:Protein Denaturation; D011506:Proteins / Q000032:analysis*
D001426:Bacterial Proteins; D004220:Disulfides; D011506:Proteins; C017079:mauC protein, Methylobacterium extorquens; D003553:Cystine; D003545:Cysteine