Recombinant Human Cyclophilin A Assay

1. Dilute Calcineurin to a concentration of 1.17 µg/mL in assay buffer.
2. Dilute Calmodulin to a concentration of 16.8 µg/mL (1 µM) in assay buffer.
3. Make serial dilutions of Cyclophilin A in assay buffer to the following pre-incubation concentrations: 10000, 5000, 2500, 1250, 625, 313, 156, 78, 39, and 20 nM.
4. Dilute Cyclosporin A to 30 µM in assay buffer.
5. Add the following to the wells of a clear plate:

   Samples	Positive Control (prepare two)	Negative Control (prepare one)
   25 µL of Calcineurin	25 µL of Calcineurin	25 µL of Calcineurin
   5 µL of Cyclophilin A at pre-incubation conc.	5 µL of assay buffer	5 µL of EDTA
   5 µL of Calmodulin	5 µL of Calmodulin	5 µL of Calmodulin
   5 µL of Cyclosporin A	5 µL of Cyclosporin A	5 µL of Cyclosporin A

6. Cover the plate, tap to mix and then incubate the plate at room temperature for 1 hour.
7. After incubation, remove the plate cover and add 10 µL of 1 mM Serine/Threonine Phosphatase Substrate I.
8. Place the cover back over plate and incubate at 37° C with shaking for 1 hour.
9. During the second incubation, prepare phosphate standard curve. Prepare serial dilutions in assay buffer at the following concentrations: 100, 50, 25, 12.5, 6.25, 3.13, and 1.57 nM.
10. Add 50 µL of the phosphate curve to the plate, in duplicate, including a 0 mM phosphate point (50 µL assay buffer).
11. Add 10 µL of malachite green reagent A to each well, tap to mix, and incubate at RT for 10 minutes.
12. Add 10 µL of malachite green reagent B to each well, tap to mix, and incubate at RT for 20 minutes.
13. Read at 620 nM to determine the phosphate liberated from the samples by using the standard curve.
14. Plot Cyclophilin A concentration (nM) vs. Phosphate released (nmoles) to determine an IC₅₀ value.

Representative Data – Figure at right

Note: Some differences from protocol described above.

The concentration of Cyclosporin A was fixed at 3 µM and the concentration of Cyclophilin in each well ranged between 3 and 1000 nM.

Red square: phosphate released in the absence of Cyclophilin A or Cyclosporin A; Blue square: phosphate released in the presence of 10 mM EDTA; Diagonal cross: phosphate released with 1 mM Cyclophilin A added but without Cyclosporin A; Open circles: phosphate released in the presence of 3 µM Cyclosporin A and the indicated concentrations of Cyclophilin A.