A procedure for the detection of free thiol-containing proteins on a polyvinylidene difluoride membrane.
Bovine serum albumin (BSA), which has a free thiol, was blotted onto a polyvinylidene difluoride membrane. The membrane was reacted with a sulfhydryl-reactive (maleimide-containing) biotin derivative, 1-biotinamido-4-[4'-(maleimidomethyl) cyclohexanecarboxamido] butane (Biotin-BMCC), and then probed. BSA on membranes was detected semi-quantitatively at 50 ng of protein (0.76 pmol of free thiol) and among range of higher extent. BSA on membranes was less efficiently biotinylated compared with biotinylation in solution. Regardless, these results suggested that sulfhydryl-containing proteins were specifically and semi-quantitatively identified by membrane biotinylation with Biotin-BMCC.
Kamiya
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Central Research Laboratories, Green Cross Corporation, Osaka, Japan. kamiya@greencross.co.jp
D000327:Adsorption; D000818:Animals; D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western; D002417:Cattle; D007118:Immunoassay / Q000379:methods*; D007202:Indicators and Reagents; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000032:analysis*; D005465:Fluorocarbon Polymers
D001710:Biotin; D007202:Indicators and Reagents; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins; D012710:Serum Albumin, Bovine; D013438:Sulfhydryl Compounds; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers