Some problems arising in SDS-PAGE and silver staining of reduced protein samples

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  • Senda Harumi
    Department of Biochemistry, Faculty of Science, Niigata University
  • Hasegawa Kazuko
    Department of Biochemistry, Faculty of Science, Niigata University
  • Sugano Hiroshi
    Department of Biochemistry, Faculty of Science, Niigata University

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  • 還元蛋白試料のSDS-PAGE-銀染色の際に生ずる問題点

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The presence of 2-mercaptoethanol or dithiothreitol in the protein denaturing solution used to prepare samples for SDS-PAGE gives rise to artifacts by silver staining of the gel. Addition of iodoacetamide to the reduced protein sample prevents the appearance of artifacts. Silver staining is strengthened by the dithiothreitol treatment but weakened by subsequent treatment with iodoacetamide. High concentration of dithiothreitol also inhibited development of silver staining. These effects of iodoacetamide or high concentration of dithiothreitol were observed more pronouncedly with the silver-staining method of Merril et al.4) than of Guevara.5) Mobility of the protein having intramolecular disulfide bonds is more or less slowed down depending on the concentration of dithiothreitol used. From these results it is concluded that the most suitable concentration of dithiothreitol is in the range of 40 to 80mM from the viewpoints of sensitivity of silver staining, completion of mobility shift, and sharpness of developed protein bands.

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