Calcineurin, also called Protein Phosphatase 2B, PP2B, PPP2B, Protein Phosphatase 3, and PPP3, is an enzyme that dephosphorylates serine and threonine residues in proteins. It is a heterodimer of a 59,000 dalton catalytic A subunit and a 19,000 dalton regulatory B subunit that is activated by the binding of calcium ions and calmodulin (1). Calcineurin is expressed in many tissues, but its levels are highest in the brain, where it may play a role in learning and memory (2). It has many substrates, including NFAT, a transcription factor that is activated by dephosphorylation (3). Complexes of the immunosuppressants cyclosporin and FK506 with immunophilin proteins such as cyclophilin and FKBP12 are potent and specific inhibitors of Calcineurin activity (4). Alterations in Calcineurin activity are suspected to play a role in cardiac hypertrophy (5) and graft versus host disease in organ transplantation (6).
Recombinant Human Calcineurin Protein, CF
R&D Systems | Catalog # 3160-CA
Discontinued Product
3160-CA has been discontinued.
View all Calcineurin products.
Key Product Details
- R&D Systems Sf 21 (baculovirus)-derived Recombinant Human Calcineurin Protein (3160-CA)
- Quality control testing to verify active proteins with lot specific assays by in-house scientists
- All R&D Systems proteins are covered with a 100% guarantee
Source
Sf 21 (baculovirus)
Accession Number
Applications
Enzyme Activity
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Product Specifications
Source
Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Calcineurin protein
| Human Calcineurin A (Ser2-Gln521) Accession # NP_000935 |
|
| Human Calcineurin B (Met1-Val170) Accession # NP_000936 |
|
| N-terminus | C-terminus |
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
N-terminal Sequence Analysis
Asp15 (Calcineurin A) & Asp17 (Calcineurin B)
Predicted Molecular Mass
57 kDa (Calcineurin A), 18 kDa (Calcineurin B)
SDS-PAGE
43-65 kDa & 15-20 kDa, reducing conditions
Activity
Measured by its ability to dephosphorylate the peptide substrate, DLDVPIPGRFDRRVS(PO3)VAAE(Catalog # ES012).
The specific activity is >900 nmol/min/mg, under the described conditions.
The specific activity is >900 nmol/min/mg, under the described conditions.
Formulation, Preparation, and Storage
3160-CA
| Formulation | Supplied as a 0.2 μm filtered solution in HEPES, CaCl2, MgCl2, DTT and Glycerol. |
| 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.
|
Background: Calcineurin
References
- Stemmer, P.M and C.B. Klee (1994) Biochemistry 33:6859.
- Zeng H. et al. (2001) Cell 107:617.
- Okamura, H. et al. (2000) Molecular Cell 6:539.
- Liu, J. et al. (1992) Biochemistry 31:3896.
- Molkentin, J.D. (2000) Circulation Research 87:731.
- Sanquer, S.A. et al. (2004) Transplantation 77:854.
Alternate Names
CALN
Entrez Gene IDs
5530 (Human)
Gene Symbol
PPP3CA
Additional Calcineurin Products
Product Documents for Recombinant Human Calcineurin Protein, CF
Certificate of Analysis
To download a Certificate of Analysis, please enter a lot or batch number in the search box below.
Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Recombinant Human Calcineurin Protein, CF
For research use only
Citations for Recombinant Human Calcineurin Protein, CF
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Protocols
View specific protocols for Recombinant Human Calcineurin Protein, CF (3160-CA):
Materials
- Assay Buffer: 20 mM Tris, 10 mM MgCl2, 0.1 mM CaCl2, 1 mg/mL BSA, pH 7.5
- Recombinant Human Calcineurin (rhCalcineurin) (Catalog # 3160-CA)
- Substrate: Serine/Threonine Phosphatase substrate I (Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser(PO3)-Val-Ala-Ala-Glu) (Catalog # ES012), 10 mM stock in deionized water
- Calmodulin (Sigma, Catalog # P1431), 0.168 mg/mL stock in Assay Buffer
- EDTA (Sigma, Catalog # E6758), 0.1 M stock in deionized water (pH 8.0)
- Incubator at 37 °C able to shake samples
- Malachite Green Phosphate Detection Kit (Catalog # DY996)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Sealers (Corning, Catalog # 3095)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhCalcineurin to a concentration of 0.71 µg/mL in Assay Buffer. Add 35 µL to each well.
- Dilute Calmodulin to a concentration of 16.8 µg/mL (1 µM) in Assay Buffer. Add 5 µL to each well. As a control add 5 µL EDTA instead of Calmodulin to fully inhibit rhCalcineurin.
- Cover plate, tap to mix and incubate plate at room temperature for 1 hour.
- After incubation remove plate cover and add 10 µL of 1 mM Substrate.
- Place cover back over plate and incubate at 37 °C with shaking for 30 min.
- During 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 µM.
- At the end of the incubation, quickly add 50 µL of the phosphate curve to the plate, in duplicate, including a 0 µM phosphate point (50 μL Assay Buffer).
- Add 10 µL of Malachite Green Reagent A, tap to mix, and incubate at room temperature for 10 minutes.
- Add 10 µL of Malachite Green Reagent B, tap to mix, and incubate at room temperature for 20 minutes.
- Read at 620 nm and determine phosphate liberated from samples using standard curve.
- Calculate the specific activity:
| Specific Activity (nmol/min/mg) = | Phosphate released* (nmol) |
| Incubation time (min) x amount of enzyme (mg) |
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Substrate Blank. Per Well:
- Phosphate standard curve: 5.0, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, and 0 nmol
- Calcineurin: 0.00002485 mg
