Recombinant Human FKBP12 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 FKBP12 in assay buffer to the following pre-incubation concentrations: 10000, 2500, 625, 313, 156, 78, 39, 9.8, 2.4 and 0.61 nM.
4. Dilute FK506 to 30 µM in assay buffer.
5. Add the following to the wells of a clear plate:
   [Adding in the order specified allows the largest volume to mix all four components together]

   Samples	Positive Control (prepare two)	Neg. Control (optional, prepare one)
   5 µL of FKBP12 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 FK506	5 µL of FK506	5 µL of FK506
   25 µL of Calcineurin	25 µL of Calcineurin	25 µL of Calcineurin

6. Seal the plate, tap to mix and then incubate the plate at room temperature for 1 hour.
7. After incubation, remove the plate seal and add 10 µL of 1 mM Serine/Threonine Phosphatase Substrate I.
8. Reseal the 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. To every well, add 10 µL of malachite green reagent A, tap to mix, and incubate at RT for 10 minutes.
12. To every well, add 10 µL of malachite green reagent B, 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 FKBP12 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 FK506 was fixed at 3 µM and the concentration of FKBP12 in each well ranged between 0.061 and 1000 nM.

Open circle: phosphate released in the absence of FKBP12; open triangle: phosphate released in the presence of 10 mM EDTA; filled circles: phosphate released in the presence of 3 µM FK506 and the indicated concentrations of FKBP12.