>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
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.
41 kDa, reducing conditions
Read Publication using 4150-PP in the following applications:
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.
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
phosphatase 2C alpha
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
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.
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