Antithrombin III/Serpin C1 Protein, Human (HEK293, His)

Materials
Assay buffer: 50 mM Tris, 10 mM CaCl?, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
Thrombin (MW 37kDa)（HY-114164）
Antithrombin III/Serpin C1 Protein, Human (HEK293, His)（HY-P72831）
Heparin (HY-17567A), diluted to 20 mg/mL with deionized water
Substrate: BOC-Val-Pro-Arg-AMC, 10 mM dissolved in DMSO

Procedure
1. Dilute Thrombin to 0.5 μg/mL with assay buffer (final heparin concentration of 48.6 μg/mL) .
2. Prepare different concentrations of rhSerpin C1 with assay buffer, and perform the following serial dilutions: 50, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078, 0.039 nM.
3. Mix equal volumes of Human Serpin C1 with Thrombin/Heparin (from step 1) ; for the control group, add equal volumes of assay buffer and Thrombin/Heparin.
4. Incubate the mixtures at room temperature for 30 minutes.
5. After incubation, dilute the mixtures 1/5 with assay buffer.
6. Dilute the substrate to 200 μM with assay buffer.
7. In a 96-well plate, add 50 μL of the diluted reaction mixture and 50 μL of 200 μM substrate to initiate the reaction.
8. Read in kinetic mode for 5 minutes with excitation and emission wavelengths at 380 nm and 460 nm (top read) .
9. Determine the IC₅₀ of Human Serpin C1 by fitting the RFU/min versus concentration curve with a 4-parameter logistic (4-PL) model.