Human Antithrombin III protein (active)

The activity of recombinant human antithrombin III was measured by its ability to inhibit thrombin cleavage of a fluorogenic peptide substrate Boc-VPR-AMC in the assay buffer 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5. Thrombin was diluted to 0.5 U with heparin at 50 μg/ml in the assay buffer and 10 ul different concentrations of recombinant human antithrombin III (MW: 50 KD) was incubated with 10 ul diluted thrombin at 37 ºC for 30 minutes. Loading 50 μL of the incubated mixtures which were diluted five-fold in assay buffer into empty wells of a plate, and start the reaction by adding 50 μL of 200 μM substrate. Include a substrate blank containing 50 μL of assay buffer and 50 μL of 200 μM substrate. Then read at excitiation and emission wavelengths of 380 nm and 460 nm, respectively, in kinetic mode for 5 minutes. This recombinant human antithrombin III significantly decreased thrombin activity. The inhibition IC₅₀ was <8 nM.