Transblot studies with biotin-labeled proteins: electrophoretic mobilities and detection limits.
Molecular weight standard proteins, mouse IgG as well as several antigens cross-reacting with the carcinoembryonic antigen (CEA), were biotin labeled, submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. The bound proteins were revealed by the use of avidin-peroxidase conjugates and a suitable substrate. The ratio of N-hydrosuccinimido biotin (NHSB) to protein yielding the lowest detection limit was determined. At an optimal NHSB/protein ratio, 0.33 ng of IgG heavy chains and 0.17 ng of IgG light chains could be visualized. With the exception of human albumin and ovalbumin, the increase in apparent molecular weight after biotin labeling was less than 10% for the proteins tested. The method has proven to be a valuable addition to Western blots performed with CEA-related antigens and monoclonal anti-CEA antibodies.
D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives*; D002272:Carcinoembryonic Antigen / Q000032:analysis; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D007074:Immunoglobulin G / Q000032:analysis; D051379:Mice; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis*; D013194:Staining and Labeling; D013388:Succinimides
D002272:Carcinoembryonic Antigen; D007074:Immunoglobulin G; D011506:Proteins; D013388:Succinimides; C033788:biotinyl N-hydroxysuccinimide ester; D001710:Biotin; D003101:Collodion