Recombinant Human PP2C alpha/PPM1A Protein, CF Summary
Product Specifications
Gly2-Lys324, with a C-terminal 6-His tag
Analysis
Product Datasheets
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.
4150-PP
| Formulation | Supplied as a 0.2 μm filtered solution in HEPES, DTT, NaCl and MnCl2. |
| 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: 20 mM Tris, 10 mM MgCl2, 1 mg/mL BSA, 0.02% (w/v) Brij-35, pH 7.5
- Recombinant Human PP2C alpha /PPM1A (rhPP2C alpha ) (Catalog # 4150-PP)
- Substrate: Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser(PO3)-Val-Ala-Ala-Glu (Catalog # ES012)
- EDTA (Sigma, Catalog # E-4884), 0.5 M stock in deionized water
- Malachite Green Phosphate Detection Kit (Catalog # DY996)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Incubator (37 °C) with orbital shaking or capable of containing an orbital plate shaker
- Dilute rhPP2C alpha to 0.75 µg/mL in Assay Buffer.
- Load in plate 40 µL per well of 0.75 µg/mL rhPP2C alpha (active wells). Include inactivated control wells by adding 5 µL of 0.1 M EDTA (inhibits rhPP2C alpha activity) to 40 µL of 0.75 µg/mL rhPP2C alpha. To the active wells add 5 µL of Assay Buffer instead of EDTA. Also load 45 µL of Assay Buffer to wells to be used as Substrate Blanks.
- Tap to mix, seal plate and incubate at room temperature for 10 minutes.
- Prepare a standard curve by adding 10 μL of the 1 M Phosphate Standard to 990 μL of Assay Buffer for a 10 mM stock.
- Continue by adding 10 μL of the 10 mM phosphate stock to 990 μL of Assay Buffer for a 100 μM stock (this is the first dilution to use as a standard).
- Perform six additional one-half serial dilutions of the 100 μM Phosphate Standard stock. The standard curve has a range of 0.086 to 5.5 nmol per well.
- Load 55 μL of the standard curve to empty wells. Include curve blanks containing 55 μL of Assay Buffer.
- Dilute Substrate to 1 mM in Assay Buffer.
- Add 10 μL of the diluted Substrate to the active, inactivated and Substrate Blank wells.
- Seal the plate and incubate at 37 °C for 30 minutes with shaking.
- After incubation, add 10 μL of Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
- Add 10 μL of Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- Calculate 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 Reaction:- rhPP2C alpha : 0.00003 mg
- Substrate: 0.182 mM
- Phosphate Curve: 5.5, 2.75, 1.375, 0.688, 0.344, 0.172, 0.086 nmol
Reconstitution Calculator
Background: PP2C alpha/PPM1A
Protein phosphatase 2C alpha, also called PP2C alpha, Protein Phosphatase, Magnesium-dependent type 1A, and PPM1A, dephosphorylates proteins with some preference for phospho‑threonine over phospho‑serine residues (1). It is insensitive to the PP1 and PP2a inhibitors such as okadaic acid, (2) but has an absolute requirement for either magnesium or manganese ions (3) and can be activated by unsaturated fatty acids such as arachidonic and oleic acids (4). Overexpression of PP2C alpha causes G2/M cell cycle arrest and apoptosis that has been associated with the activation of p53 (5). Dephosphorylation of proteins in the nucleus by PP2C alpha is suspected to play a role in terminating or reducing the sensitivity of the responses to SMADS (6) and stress-activated proteins such as p38 and JNK (7).
- Donella-Deanna, A. et al. (1991) Biochimica et Biophysica Acta 1094:130.
- Bialojan, C. and A. Takai (1998) Biochem. J. 256:283.
- Cohen, P. Annu. Rev. Biochem. (1989) 58:453.
- Klumpp, S. et al. (1998) FEBS Let. 437:229.
- Ofek, P. et al. (2003) J. Biol. Chem. 278:14299.
- Lin, X. et al. (2006) Cell 125:915.
- Takekawa, M. et al. (1998) EMBO J. 17:4744.
Citation for Recombinant Human PP2C alpha/PPM1A 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.
1 Citation: Showing 1 - 1
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Kaposi's sarcoma-associated herpesvirus induces rapid release of angiopoietin-2 from endothelial cells.
Authors: Ye F, Zhou F, Nithianantham S, Chandran B, Yu X, Weinberg A, Gao S
J Virol, 2013-03-27;87(11):6326-35.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay
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