Recombinant Human FKBP12 Protein, CF Summary
Product Specifications
Gly2-Glu108, with an N-terminal Met and 6-His tag
Analysis
Product Datasheets
3777-FK
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
3777-FK
| Formulation | Supplied as a 0.2 μm filtered solution in HEPES and NaCl. |
| Shipping | The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- 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.
- Prepare Assay Buffer and place on ice.
- Prepare 20 mg/mL Lithium Chloride in Trifluoroethanol.
- 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.
- 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.
- Place micro-cuvette on ice for 10 minutes.
- Quickly add 10 µL of cold 3 mM Suc-AAPF-pNA. Invert vigorously 1-2 seconds and wipe condensation off micro-cuvette.
- Read immediately at an absorbance of 405 nm for 1 minute in kinetic mode.
- Clean micro-cuvette thoroughly with ethanol and cold deionized water.
- Dilute rhFKBP12 to 100 µg/mL in cold Assay Buffer.
- 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.
- Place micro-cuvette on ice for 10 minutes.
- Quickly add 10 µL of cold 3 mM Suc-AAPF-pNA. Invert vigorously 1-2 seconds and wipe condensation off micro-cuvette.
- Read immediately at an absorbance of 405 nm for 1 minute in kinetic mode.
- Clean micro-cuvette thoroughly with ethanol and cold deionized water.
- Use the first 30 seconds of the reads to determine the test rate.
|
Specific Activity (pmol/min/µg) = |
Adjusted Rate* (OD/min) x well volume (L) x 1012 pmol/mol |
| ext. coeff** (M-1cm-1) x path length*** (cm) x amount of enzyme (µg) |
*Adjusted for Blank
**Using the extinction coefficient 9300 M-1cm-1
***Using the path length 1 cm
Note: the output of many spectrophotometers is in mOD. Per Reaction:
- rhFKBP12: 1 µg
- Suc-AAPF-pNA: 150 µM
- Chymotrypsin: 1.2 mg
Background: FKBP12
FK506 binding protein, 12 kilodalton molecular weight (FKBP12), also called FKBP1, was originally characterized as a peptidyl-prolyl isomerase that catalyzes the transition between cis- and trans-proline residues critical for proper folding of proteins. The macrolide immunosuppressants FK506 (Tacrolimus) and rapamycin bind to FKBP12 with high affinity, while the structurally related compound cyclosporine binds with a much lower affinity (1). The binding of these drugs causes FKBP12 to become a potent inhibitor of calcineurin phosphatase activity (2) and TOR kinase activity (3). The inhibition of protein phosphatase activity is highly selective for calcineurin (2), making the FK506/FKBP12 complex a useful tool in the study of this enzyme. Knockout mice lacking FKBP12 are morphologically normal, but develop cardiomyopathies that may be related to dysregulation of ryanodyne receptors (4).
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