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Enzyme electrophoresis method in analysis of active components of haemostasis system

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Author(s): Ludmila Ostapchenko | Oleksiy Savchuk | Nataliia Burlova-Vasilieva

Journal: Advances in Bioscience and Biotechnology
ISSN 2156-8456

Volume: 02;
Issue: 01;
Start page: 20;
Date: 2011;
Original page

Keywords: Substrate-Containing Electrophoresis | Enzyme Electrophoresis | Haemostasis | Proteins Activity | Proteins Identification

ABSTRACT
The novel modifications of substrate-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis that can be used for the detection of proteases and its activators are reported. The protease/activator samples were separated on a protein substrate-SDS-polyacrylamide gel. To detect plasminogen activators fibrinogen and Glu-plasminogen were incorporated into the SDS-PAG followed by 1 h incubation at 37?C in thrombin solution (1 NIH/ml). After electrophoresis the gel was stained according to the standard protocol. To detect fibrin-unspecific plasminogen activators from snake venom incubation in thrombin solution was substituted for 12 h incubation in 50 mM Tris-HCl (pH 7.4). To detect fibrinogen-degrading enzymes fibrinogen-containing gel was used. Activity of protease/activator was visualized in the gel as clear bands against the dark background. These new techniques offer several advantages including determination of the quantity and activity of t-PA and urokinase, however cannot be recommended for precise quantification of activators; the total procedure is quite quick and simple; method is convenient tool for detection of novel protein-protein interactions in haemostasis system; the sensitivity of the method is ≤0.01 IU per track.

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