Recombinant Human PP2C alpha/PPM1A Protein, CF

R&D Systems | Catalog # 4150-PP

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Key Product Details

  • R&D Systems E. coli-derived Recombinant Human PP2C alpha/PPM1A Protein (4150-PP)
  • 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

E. coli

Accession Number

NP_808820

Applications

Enzyme Activity
View all PP2C alpha/PPM1A Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human PP2C alpha/PPM1A protein
Gly2-Lys324, with a C-terminal 6-His tag

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.

N-terminal Sequence Analysis

Gly2

Predicted Molecular Mass

37 kDa

SDS-PAGE

41 kDa, reducing conditions

Activity

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.

Formulation, Preparation, and Storage

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

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).

References

  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.

Long Name

Protein Phosphatase 2C alpha

Alternate Names

MMPa-2, MPPa-1, Pp2c1, PPM1A

Entrez Gene IDs

5494 (Human); 19042 (Mouse); 24666 (Rat)

Gene Symbol

PPM1A

UniProt

NP_808820

Additional PP2C alpha/PPM1A Products

Product Documents for Recombinant Human PP2C alpha/PPM1A Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human PP2C alpha/PPM1A Protein, CF

For research use only

Citations for Recombinant Human PP2C alpha/PPM1A Protein, CF

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Protocols

View specific protocols for Recombinant Human PP2C alpha/PPM1A Protein, CF (4150-PP):

Materials
  • 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

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