Recombinant Human Cyclophilin A Protein, CF

• Assay Buffer (AB): 20 mM Tris, 10 mM MgCl₂, 0.1 mM CaCl₂, 1 mg/mL BSA, pH 7.5
• Recombinant Human Cyclophilin A (rhCyclophilin A) (MW: 18 kDa) (Catalog # 3589-CAB)
• Recombinant Human Calcineurin (rhCalcineurin) (Catalog # 3160-CA)
• Substrate: Serine/Threonine Phosphatase Substrate I (Catalog # ES012), 10 mM stock in deionized water
• Calmodulin (Sigma, Catalog # P1431), 0.168 mg/mL stock in Assay Buffer
• EDTA (Sigma, Catalog # E6758), 0.1 M stock in deionized water (pH to 8.0)
• Cyclosporin A (Sigma, Catalog # C1832), 1 mM stock in DMSO
• Incubator at 37 °C able to shake microplate
• Malachite Green Phosphate Detection Kit (Catalog # DY996)
• 96-well Clear Plate (Catalog # DY990)
• Plate Sealers (Corning, Catalog # 3095)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute Calcineurin to 1.17 µg/mL, Calmodulin to 16.8 µg/mL (1 µM), and Cyclosporin A to 30 µM in Assay Buffer (AB).
2. Dilute Cyclophilin A in AB to the following concentrations: neat, 10000, 5000, 2500, 1250, 625, 313, 156, 78, 39, and 20 nM.
3. Add the following to the wells of a plate (add largest volume last to mix all together):

   Samples                                                5 µL of Cyclophilin A dilutions 5 µL of Calmodulin 5 µL of Cyclosporin A 25 µL of Calcineurin

   Pos. Control (prepare two)         5 µL of assay buffer 5 µL of Calmodulin 5 µL of Cyclosporin A 25 µL of Calcineurin

   Neg. Control (optional, prepare one) 5 µL of EDTA 5 µL of Calmodulin 5 µL of Cyclosporin A 25 µL of Calcineurin

4. Seal plate, tap to mix and incubate plate at room temperature for 1 hr.
5. After incubation remove seal and add 10 µL of 1 mM Substrate.
6. Reseal and incubate at 37 °C with shaking for 1 hr.
7. During second incubation, prepare kit standard curve. Prepare serial dilutions in AB at the following conc.: 100, 50, 25, 12.5, 6.25, 3.13, and 1.57 µM.
8. Add 50 µL of the phos. curve to the plate, in duplicate, including a 0 mM phos. point (50 µL AB).
9. To all wells add 10 µL of kit reagent A, tap to mix, and incubate at RT for 10 min.
10. To all wells add 10 µL of kit reagent B, tap to mix, and incubate at RT for 20 min.
11. Read at 620 nm.
12. Determine the 50% inhibiting conc. (IC₅₀) of rhCyclophilin A by plotting conc. (nM) vs. Phos. released (nmol) with 4-PL fitting.
13. The specific activity of rhCalcineurin at each point can be determined using the following eqn. (if needed):

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Negative Control.

Per Well:

• Phos. standard: 5.0, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, and 0 nmol
• rhCyclophilin A: nt/14, 714, 357, 179, 89, 45, 22, 11, 5.6, 2.8, 1.4, 0.0 nM 
• rhCalcineurin: 0.00002925 mg