Effect of β-Mercaptoethanol on the Detection of Biotinylated Proteins

✍ Scribed by S.A. Weston; B. Crossett; D.S. Tuckwell; M.J. Humphries

• Book ID: 102967375
• Publisher: Elsevier Science
• Year: 1995
• Tongue: English
• Weight: 597 KB
• Volume: 225
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1995.1103

✦ Synopsis

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-horseradish 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. 1995 Academic Press, Inc.