The Thrombin Inhibitor Screening Assay is a colorimetric assay designed to measure the activity of human alpha thrombin for screening and profiling applications. The assay kit comes in a convenient 96-well or 384-well format and contains enough purified human alpha thrombin, a chromogenic substrate, and PR-02 buffer for 100 or 400 reactions.

To determine the effect of an inhibitor on Thrombin activity, the enzyme should be preincubated with or without the test inhibitor prior to adding the chromogenic substrate to the reaction. The assay was functionally validated using Dabigatran, a potent inhibitor of thrombin.

Figure 1: Illustration of the assay principle.
Upon proteolysis, thrombin cleaves the chromogenic substrate at the C-terminal end releasing p-nitroanilid (pNA), which produces a yellow color that is measurable photometrically at λ=405 nm. The increase in color is proportional to thrombin activity.