Recombinant Human FKBP12 Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Inhibition Activity
Format
Carrier-Free

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Recombinant Human FKBP12 Protein, CF Summary

Details of Functionality
Measured by its ability to convert the substrate, Suc-AAPF-pNA, from Cis to Trans formation. The specific activity is >2500 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived human FKBP12 protein
Gly2-Glu108, with an N-terminal Met and 6-His tag
Accession #
N-terminal Sequence
Met
Protein/Peptide Type
Recombinant Proteins
Gene
FKBP1A
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Dilutions
  • Inhibition Activity
Theoretical MW
13 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
13 kDa, reducing conditions

Packaging, Storage & Formulations

Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in HEPES and NaCl.
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
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.

  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.

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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human FKBP12 Protein, CF

  • 12 kDa FKBP
  • EC 5.2.1.8
  • FK506 binding protein 1A, 12kDa
  • FK506-binding protein 1A
  • FKBP1
  • FKBP12
  • FKBP12C
  • FKBP12FKBP12-Exip3
  • FKBP-12T-cell, 12-kD
  • FKBP1a
  • FKBP-1A
  • peptidyl-prolyl cis-trans isomerase FKBP1A
  • PKC12
  • PPIase FKBP1A
  • PPIASE
  • protein kinase C inhibitor 2
  • rotamase

Background

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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Bioinformatics

Gene Symbol FKBP1A
Uniprot