Amidolytic detection of prothrombin activation products after SDS-gel electrophoresis
Tans, G.; Janssen-Claessen, T.; Rosing, J.
Thrombosis and Haemostasis 61(3): 386-391
1989
ISSN/ISBN: 0340-6245 PMID: 2799753 Document Number: 339270
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).