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  • Title: Effect of beta-mercaptoethanol on the detection of biotinylated proteins.
    Author: Weston SA, Crossett B, Tuckwell DS, Humphries MJ.
    Journal: Anal Biochem; 1995 Feb 10; 225(1):28-33. PubMed ID: 7778784.
    Abstract:
    Biotinylated proteins were visualized by enhanced chemiluminescence (ECL) or conventional autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein transfer onto nitrocellulose. Soaking polyacrylamide gels run under nonreducing conditions in beta-mercaptoethanol (2-ME) prior to protein transfer onto nitrocellulose resulted in a 2- to 10-fold augmentation of the resultant signal. This enhancement was observed for both disulfide- and nondisulfide-bonded proteins. Furthermore, 2-ME had no effect on either the activity of the extravidin-horse-radish peroxidase conjugate, used to detect biotin moieties, or the net protein transfer onto nitrocellulose. Thus, we propose that amplification of either ECL or gamma emission following 2-ME treatment is due to its ability to modify protein conformation, which in turn provides greater access of avidin to biotin.
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