Effect of beta-mercaptoethanol on the detection of biotinylated proteins.
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.
Weston
S A
SA
School of Biological Sciences, University of Manchester, United Kingdom.
Crossett
B
B
Tuckwell
D S
DS
Humphries
M J
MJ
D001710:Biotin; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D005354:Fibrosarcoma; D006801:Humans; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D008163:Luminescent Measurements; D008623:Mercaptoethanol; D009363:Neoplasm Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis; D014407:Tumor Cells, Cultured
D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D009363:Neoplasm Proteins; D011506:Proteins; D012710:Serum Albumin, Bovine; D008623:Mercaptoethanol; D001710:Biotin