Amidolytic detection of prothrombin activation products after SDS-gel electrophoresis.
In this paper we report a method via which enzymatically active products formed during prothrombin activation can be detected by simple photographic means after SDS-gel electrophoresis, blotting onto a nitrocellulose membrane and visualization with the chromogenic substrate, S2238. After amidolytic detection the same nitrocellulose membrane can also be used for immunologic detection of prothrombin activation products, thus allowing a complete description of product formation during prothrombin activation. The detection limit of the so-called "amidoblot" is approximately 3 ng thrombin per gel sample which is comparable to the sensitivity of immunoblotting. It is further shown that the amidoblot technique can also be applied to other coagulation factors for which a suitable chromogenic substrate is available (factor XIIa, kallikrein, factor XIa, factor Xa, plasmin and activated protein C).
Tans
G
G
Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.
Janssen-Claessen
T
T
Rosing
J
J
D001780:Blood Coagulation Tests / Q000379:methods; D002384:Catalysis; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D010209:Paper; D012967:Sodium Dodecyl Sulfate; D013917:Thrombin / Q000302:isolation & purification / Q000378:metabolism*
D012967:Sodium Dodecyl Sulfate; D003101:Collodion; D013917:Thrombin