Enzyme electrophoresis method in analysis of active components of haemostasis system

  • Ostapchenko L
  • Savchuk O
  • Burlova-Vasilieva N
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Abstract

The novel modifications of substrate-containing so-dium dodecyl sulfate-polyacrylamide gel electropho-resis that can be used for the detection of proteases and its activators are reported. The protease/acti-vator samples were separated on a protein substrate-SDS-polyacrylamide gel. To detect plasminogen acti-vators fibrinogen and Glu-plasminogen were incor-porated into the SDS-PAG followed by 1 h incubation at 37˚C in thrombin solution (1 NIH/ml). After elec-trophoresis the gel was stained according to the standard protocol. To detect fibrin-unspecific plasmi-nogen 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 in-cluding determination of the quantity and activity of t-PA and urokinase, however cannot be recommended for precise quantification of activators; the total pro-cedure is quite quick and simple; method is conven-ient tool for detection of novel protein-protein inter-actions in haemostasis system; the sensitivity of the method is ≤0.01 IU per track.

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Ostapchenko, L., Savchuk, O., & Burlova-Vasilieva, N. (2011). Enzyme electrophoresis method in analysis of active components of haemostasis system. Advances in Bioscience and Biotechnology, 02(01), 20–26. https://doi.org/10.4236/abb.2011.21004

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