Recombinant Human PP2C alpha/PPM1A Protein, CF

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

Recombinant Human PP2C alpha/PPM1A Protein, CF Summary

• Details of Functionality: Measured by its ability to dephosphorylate the peptide substrate, DLDVPIPGRFDRRVS(PO₃)VAAE(Catalog # ES012). The specific activity is >400 nmol/min/mg, as measured under the described conditions.
• Source: E. coli-derived human PP2C alpha/PPM1A protein
  Gly2-Lys324, with a C-terminal 6-His tag
• Accession #: NP_808820
• N-terminal Sequence: Gly2
• Protein/Peptide Type: Recombinant Enzymes
• Gene: PPM1A
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 37 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: 41 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, DTT, NaCl and MnCl2.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• 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
  1. Dilute rhPP2C alpha to 0.75 µg/mL in Assay Buffer.
  2. 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.
  3. Tap to mix, seal plate and incubate at room temperature for 10 minutes.
  4. 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.
  5. 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).
  6. 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.
  7. Load 55 μL of the standard curve to empty wells. Include curve blanks containing 55 μL of Assay Buffer.
  8. Dilute Substrate to 1 mM in Assay Buffer.
  9. Add 10 μL of the diluted Substrate to the active, inactivated and Substrate Blank wells.
  10. Seal the plate and incubate at 37 °C for 30 minutes with shaking.
  11. After incubation, add 10 μL of Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
  12. Add 10 μL of Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  13. Read plate at 620 nm (absorbance) in endpoint mode.
  14. 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

Notes

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

Alternate Names for Recombinant Human PP2C alpha/PPM1A Protein, CF

• EC 3.1.3.16
• FLJ42306
• MGC9201
• MMPa-2
• MPPa-1
• phosphatase 2C alpha
• PP2C alpha
• Pp2c1
• PP2CA
• PP2Calpha
• PP2C-ALPHA
• PPM1A
• PPPM1A
• protein phosphatase 1A (formerly 2C), magnesium-dependent, alpha isoform
• protein phosphatase 1A
• protein phosphatase 2C alpha isoform
• Protein phosphatase 2C isoform alpha
• Protein phosphatase IA
• protein phosphatase, Mg2+/Mn2+ dependent, 1A

Background

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).

1. Donella-Deanna, A. et al. (1991) Biochimica et Biophysica Acta 1094:130.
2. Bialojan, C. and A. Takai (1998) Biochem. J. 256:283.
3. Cohen, P. Annu. Rev. Biochem. (1989) 58:453.
4. Klumpp, S. et al. (1998) FEBS Let. 437:229.
5. Ofek, P. et al. (2003) J. Biol. Chem. 278:14299.
6. Lin, X. et al. (2006) Cell 125:915.
7. Takekawa, M. et al. (1998) EMBO J. 17:4744.

Publications for PP2C alpha/PPM1A (4150-PP)(1)

Publication using 4150-PP

• Ye F, Zhou F, Nithianantham S, Chandran B, Yu X, Weinberg A, Gao S Kaposi's sarcoma-associated herpesvirus induces rapid release of angiopoietin-2 from endothelial cells. J Virol, 2013-03-27;87(11):6326-35. 2013-03-27 [PMID: 23536671] (Bioassay, Human)

Bioinformatics

• Gene Symbol: PPM1A
• Uniprot:
  • NP_808820()