Recombinant Human Calcineurin Protein, CF

Catalog #: 3160-CA
5 Citations Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
3160-CA-020

R&D Systems Recombinant Proteins and Enzymes

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Citations (5)

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Human Calcineurin Protein, CF Summary

Product Specifications

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.

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.

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
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

Product Datasheets

3160-CA

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

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

Assay Procedure

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

Reconstitution Calculator

Background: Calcineurin

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).

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.

Entrez Gene IDs

5530 (Human)

Alternate Names

Calcineurin; CALN

Related Research Areas

Citations for Recombinant Human Calcineurin Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

5 Citations: Showing 1 - 5
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The immunosuppressive drug cyclosporin A has an immunostimulatory function in CD8+ T cells
Authors: Wi beta feld, J;Hering, M;Ten Bosch, N;Cui, G;
European journal of immunology
Species: N/A
Sample Types: Recombinant Protein
Applications: Bioassay
Calcineurin-mediated dephosphorylation enhances the stability and transactivation of c-Myc
Authors: Masaki, T;Habara, M;Hanaki, S;Sato, Y;Tomiyasu, H;Miki, Y;Shimada, M;
Scientific reports
Species: Human
Sample Types: Recombinant Protein
Applications: Bioassay
Calcineurin regulates cyclin D1 stability through dephosphorylation at T286
Authors: T Goshima, M Habara, K Maeda, S Hanaki, Y Kato, M Shimada
Sci Rep, 2019-09-04;9(1):12779.
Species: Human
Sample Types: Revombinant Protein
Applications: Bioassay
Recruitment of calcineurin to the TCR positively regulates T cell activation
Nat. Immunol, 2016-12-12;0(0):.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay
Calcineurin increases glucose activation of ERK1/2 by reversing negative feedback.
Authors: Duan L, Cobb MH
Proc. Natl. Acad. Sci. U.S.A., 2010-12-06;107(51):22314-9.
Species: Human
Sample Types: Recombinant Protein
Applications: Bioassay