SDS-PAGE with subsequent immunoblotting of human serum EPO.

Separation of human serum EPO and rhEPO by SDS-PAGE followed by immunoblotting. Samples were stained with a biotinylated monoclonal antibody against hEPO using horseradish peroxidase–labeled streptavidin and a chemiluminescent substrate. (A) Lane 1: molecular weight (MW) markers. Lane 2: epoetin alfa (1 ng). Lane 3: human serum EPO (1 ng). (B) Lane 1: molecular weight markers. Lane 2: human serum EPO (1 ng) after incomplete digestion with PNGase F. Lane 3: epoetin alfa (5 ng) after incomplete digestion with PNGase F. (C) Lane 1: epoetin alfa (5 ng) after incomplete digestion with PNGase F. Lane 2: epoetin alfa (5 ng) after complete digestion with PNGase F. Lane 3: human serum EPO (2 ng) after complete digestion with PNGase F (⇓O-glycosylated EPO; ⇑ non–O-glycosylated EPO). Lane 4: molecular weight markers. (D) Lane 1: fully glycosylated epoetin alfa (5 ng). Lane 2: epoetin alfa (10 ng) after treatment withArthrobacter ureafaciens sialidase. Lane 3: molecular weight markers.