Vmax microtiter plate reader

The rate of conversion of FX to FXa was monitored in a purified system at 23 °C as described [22 (link)]. FVIII (1 nM) was activated to FVIIIa with α-thrombin (20 nM) for 1 min (or 15 seconds for rapidly decaying variants) in buffer containing 20 μM PSPCPE in FXa generation buffer (20 mM HEPES·HCl (pH 7.2), 100 mM NaCl, 5 mM CaCl₂, 0.01% Tween20, and 100 μg/mL BSA). Thrombin was inhibited with hirudin (10 units/mL) for 1 minute, and the resulting FVIIIa was incubated with 40 nM FIXa for 1 minute. 300 nM FX was added to the mixture, and the reaction was stopped with 100 mM EDTA after an additional minute. The final concentrations of thrombin and hirudin were 16.7 nM and 8.3 U/mL, respectively. FXa generated was determined using the FXa chromogenic substrate, Chromogenix S-2765 (0.46 mM). Reactions were monitored for 3 minutes at 385 nm using a V_max microtiter plate reader (Molecular Devices).

The rate of conversion of FX to FXa was monitored in a purified system at 23°C. Reconstituted FVIIIa (2 nM) was added to a reaction mixture of 40 nM FIXa and 20 μM PSPCPE in 20 mM HEPES pH 7.2, 100 mM NaCl, 5 mM CaCl₂, 0.01% Tween20, and 100 μg/mL BSA. After 2 minutes, 300 nM FX was added to the mixture, and the reaction was stopped with 100 mM EDTA after an additional 2 minutes. FXa generated was determined using the FXa chromogenic substrate, Pefachrome Xa (0.46 mM final concentration). Reactions were read for 3 minutes at 385 nm using a V_max microtiter plate reader (Molecular Devices).