Recombinant Human Calcineurin Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Calcineurin Protein, CF Summary

• Details of Functionality: 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.
• 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

• Accession #: NP_000935 (Calcineurin A) NP_000936 (Calcineurin B
• N-terminal Sequence: Asp15 (Calcineurin A) & Asp17 (Calcineurin B)
• Protein/Peptide Type: Recombinant Proteins
• Gene: PPP3CA
• 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:
  • Enzyme Activity
• Theoretical MW: 57 kDa (Calcineurin A), 18 kDa (Calcineurin B).
  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: 43-65 kDa & 15-20 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, CaCl₂, MgCl₂, DTT and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • 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
  1. Dilute rhCalcineurin to a concentration of 0.71 µg/mL in Assay Buffer. Add 35 µL to each well.
  2. 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.
  3. Cover plate, tap to mix and incubate plate at room temperature for 1 hour.
  4. After incubation remove plate cover and add 10 µL of 1 mM Substrate.
  5. Place cover back over plate and incubate at 37 °C with shaking for 30 min.
  6. 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.
  7. 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).
  8. Add 10 µL of Malachite Green Reagent A, tap to mix, and incubate at room temperature for 10 minutes.
  9. Add 10 µL of Malachite Green Reagent B, tap to mix, and incubate at room temperature for 20 minutes.
  10. Read at 620 nm and determine phosphate liberated from samples using standard curve.
  11. 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

Notes

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

Alternate Names for Recombinant Human Calcineurin Protein, CF

• calcineurin A alpha
• calcineurin subunit B type 1
• Calcineurin
• CALN
• CALNA
• CALNB1
• CCN1
• CNA1
• CNB1

Background

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).

1. Stemmer, P.M and C.B. Klee (1994) Biochemistry 33:6859.
2. Zeng H. et al. (2001) Cell 107:617.
3. Okamura, H. et al. (2000) Molecular Cell 6:539.
4. Liu, J. et al. (1992) Biochemistry 31:3896.
5. Molkentin, J.D. (2000) Circulation Research 87:731.
6. Sanquer, S.A. et al. (2004) Transplantation 77:854.

Publications for Calcineurin (3160-CA)(5)

Publications using 3160-CA

• Wi beta feld, J;Hering, M;Ten Bosch, N;Cui, G; The immunosuppressive drug cyclosporin A has an immunostimulatory function in CD8+ T cells European journal of immunology 2024-04-22 [PMID: 38650034] (Bioassay, N/A)
• Masaki, T;Habara, M;Hanaki, S;Sato, Y;Tomiyasu, H;Miki, Y;Shimada, M; Calcineurin-mediated dephosphorylation enhances the stability and transactivation of c-Myc Scientific reports 2023-08-12 [PMID: 37573463] (Bioassay, Human)
• T Goshima, M Habara, K Maeda, S Hanaki, Y Kato, M Shimada Calcineurin regulates cyclin D1 stability through dephosphorylation at T286 Sci Rep, 2019-09-04;9(1):12779. 2019-09-04 [PMID: 31484966] (Bioassay, Human)
• Recruitment of calcineurin to the TCR positively regulates T cell activation Nat. Immunol, 2016-12-12;0(0):. 2016-12-12 [PMID: 27941787] (Bioassay, Human)
• Duan L, Cobb MH Calcineurin increases glucose activation of ERK1/2 by reversing negative feedback. Proc. Natl. Acad. Sci. U.S.A., 2010-12-06;107(51):22314-9. 2010-12-06 [PMID: 21135229] (Bioassay, Human)

Bioinformatics

• Gene Symbol: PPP3CA
• Uniprot:
  • NP_000935 (Calcineurin A) NP_000936 (Calcineurin B()