Affinity chromatography of human factor VIII using human and rabbit antibodies to Factor VIII
Lane, J.L.; Ekert, H.; Vafiadis, A.
Thrombosis and Haemostasis 42(4): 1306-1315
1979
ISSN/ISBN: 0340-6245 PMID: 120620 Document Number: 149191
Factor VIII, purified by gel filtration on Sepharose 2B, has an 8 band multiple subunit structure, with MW ranging from 30,000-230,000, on reduction and SDS-PAGE G, 3% of VIII:C and 5% of VIII:Rag were attached to the column. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the protein, showed 2 faint bands with MW consistent with H and L chains of IgG. Similar experiments with antibody to factor VIII showed that 67-83% of VIII:C and 61-76% of VIII:Rag were attached to the column. Elution of the column with 0.25 M CaCl2 showed 10% of the applied VIII:C, but no VIII:Rag in the eluate. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the dissociated protein, showed an 8 band subunit structure similar to the reduced factor VIII.