Recombinant Human FKBP12 Protein, CF

• 6 months from date of receipt, -70 °C as supplied.
• 3 months, -70 °C under sterile conditions after opening.

• Assay Buffer: 50 mM HEPES, 100 mM NaCl, pH 8.0
• Recombinant Human FKBP12 (rhFKBP12 ) (Catalog # 3777-FK)
• Substrate: Suc-AAPF-pNA (Sigma, Catalog # S7388)
• Chymotrypsin (Sigma, Catalog # C3142), 60 mg/mL stock in 1 mM HCl
• Lithium Chloride (Sigma, Catalog # 213233)
• Trifluoroethanol (Fisher, Catalog # BP622)
• Ethanol
• Quartz Micro-Cuvette (VWR, Catalog # 414004-050) or equivalent
• Plate Reader with Cuvette Port (Model: SpectraMax Plus by Molecular Devices) or equivalent

Note: Time and temperature are critical to this assay. Execute substrate addition and reading steps as quickly as possible, and keep components on ice while not in use.

1. Prepare Assay Buffer and place on ice.
2. Prepare 20 mg/mL Lithium Chloride in Trifluoroethanol.
3. Prepare 3 mM Suc-AAPF-pNA in 20 mg/mL Lithium Chloride (step 2) and place on ice. Use 3 mM Suc-AAPF-pNA within 4 hours of preparation.
4. Combine 170 µL of cold Assay Buffer and 20 µL of cold 60 mg/mL Chymotrypsin in a clean micro-cuvette to create a Blank.
5. Place micro-cuvette on ice for 10 minutes.
6. Quickly add 10 µL of cold 3 mM Suc-AAPF-pNA. Invert vigorously 1-2 seconds and wipe condensation off micro-cuvette.
7. Read immediately at an absorbance of 405 nm for 1 minute in kinetic mode.
8. Clean micro-cuvette thoroughly with ethanol and cold deionized water.
9. Dilute rhFKBP12 to 100 µg/mL in cold Assay Buffer.
10. Combine 160 µL of cold Assay Buffer, 20 µL of cold 60 mg/mL Chymotrypsin, and 10 µL of cold 100 µg/mL rhFKBP12 in a clean micro-cuvette.
11. Place micro-cuvette on ice for 10 minutes.
12. Quickly add 10 µL of cold 3 mM Suc-AAPF-pNA. Invert vigorously 1-2 seconds and wipe condensation off micro-cuvette.
13. Read immediately at an absorbance of 405 nm for 1 minute in kinetic mode.
14. Clean micro-cuvette thoroughly with ethanol and cold deionized water.
15. Use the first 30 seconds of the reads to determine the test rate.

• rhFKBP12: 1 µg
• Suc-AAPF-pNA: 150 µM
• Chymotrypsin: 1.2 mg