Cyclophilin A Protein, Mouse

Materials
Assay buffer: 20 mM Tris, 10 mM MgCl₂, 0.1 mM CaCl₂, 1 mg/mL BSA, pH 7.5
Test protein：Cyclophilin A Protein, Mouse (HY-P70007A, CYPA)
Calcineurin Protein, Human
Calmodulin Protein, Human (HY-P7710)
Substrate: Serine/Threonine Phosphatase Substrate I, 10 mM stock solution in deionized water
Cyclosporin A, 1 mM stock solution in DMSO
Malachite Green Phosphate Assay Kit

Procedure
1. Dilute Calcineurin to 1.17 μg/mL, Calmodulin to 16.8 μg/mL, and Cyclosporin A to 30 μM in the assay buffer.
2. Dilute Human CYPA to the following concentrations: 20000, 10000, 5000, 2500, 1250, 625, 313, 156, 78, 39, and 20 nM.
3. Add the following to the microplate wells:
Sample group: 5 μL of Human CYPA dilution, 5 μL Calmodulin, 5 μL of Cyclosporin A, 25 μL of Calcineurin.
Control group: 5 μL of assay buffer, 5 μL Calmodulin, 5 μL of Cyclosporin A, 25 μL of Calcineurin.
4. Seal the plate, mix by tapping, and incubate at room temperature for 1 hour.
5. After incubation, remove the seal and add 10 μL of 1 mM substrate. The total volume should be 50 μL.
6. Re-seal the plate and incubate with shaking at 37°C for 1 hour. Adjust the volume to 200 μL with assay buffer.
7. Add 70 μL of chromogenic reagent to each well, gently mix by pipetting up and down, and allow to stand at room temperature for 30 minutes.
8. Measure at 630 nm (absorbance) in endpoint mode.
9. Determine the 50% inhibitory concentration (IC₅₀).