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Protein Phosphatase Inhibitor 2 (I-2) |
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- Type 1 protein phosphatase (PP1) specific Inhibitor
- Rabbit skeletal muscle, recombinant (E. coli)
- Supplied with 10X Reaction Buffer
Description:
Protein Phosphatase Inhibitor-2 (I-2) is a 204 amino acid heat stable protein which specifically inhibits the catalytic subunit of type 1 protein phosphatases (PP1) at nanomolar concentrations (IC50 ~ 2 nM).
I-2 can be used to distinguish type 1 from type 2 protein phosphatases, which are the major protein serine/threonine phosphatases in eurkaryotic cells (4,5).
Source:
Isolated from a strain of E. coli that carries the coding sequence for rabbit skeletal muscle I-2 polypeptide under the control of T7 expression system (kindly provided by Dr. A. A. DePaoli-Roach) (3).
Reagents Supplied:
PP1 Reaction Buffer Pack (10X)
Properties & Usage
Molecular Weight:
Theoretical: 22,800 daltons and Apparent: 31,000 daltons
Specific Activity:
1,000,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X PP1 Reaction Buffer
Supplemented with 1 mM MnCl2 and 5 mM Caffeine
Incubate at
30°C.
1X PP1 Reaction Buffer:
50 mM HEPES
100 mM NaCl
2 mM Dithiothreitol
0.1 mM EGTA
0.025 % Tween-20
pH 7.5 @ 25°C
Unit Definition:
One unit is defined as the amount of I-2 required to inhibit 0.01 unit of Protein Phosphatase 1 (PP1) by 50%. One unit of PP1 is defined as the amount of enzyme that releases 1 nmol of phosphate from phosphorylase a (10 µM) per minute at 30°C in PP1 Reaction Buffer supplemented with 1 mM MnCl2 (for recombinant PP1) and 5 mM caffeine in a 50 µl reaction (2,5).
Concentration:
1 mg/ml
Unit Assay Substrate: PP1 Reaction Buffer
Storage Conditions:
50 mM HEPES
1 mM EDTA
50% Glycerol
0.01% Brij 35
pH 7.0 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- Supplement with 1 mM MnCl2 and 5 mM caffeine (required only if phosphorylase a is the substrate).
- I-2 has been purified to > 95% homogeneity as determined by SDS-PAGE and Coomassie Blue staining.
- Recommended long term storage -70°C; avoid repeated freeze/thaw cycles.
Usage notes:- PP1 or cell extracts should be diluted so that the concentration of phosphatase activity is less than 0.1 units/ml and preincubated with I-2 for 10-15 minutes prior to assay (2,5).
- 10-100 ng of I-2 will typically inhibit > 90% of phosphatase activity of 0.02 unit recombinant PP1 (NEB #P0754) in 10 minutes in a 50 µl reaction.
- 0.1-1 µg can inhibit > 90% of PP1 phosphatase activity in cell extracts (5).
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
I-2 contains no detectable protease activity. Tests for phosphatase activity showed no detectable activity.
Protease Assay:
Incubation of 2 µg of Protein Phosphatase Inhibitor 2 (I-2) with a standardized mixture of proteins for 2 hours at 30°C, no proteolytic activity could be detected by SDS-PAGE.
PNP-Glycosidase Assay (16 hour test):
Incubation of 2 µg of Protein Phosphatase Inhibitor 2 (I-2) with 50 mM p-nitrophenyl phosphate for 2 hours at 30°C, no phosphatase activity could be detected by spectrophotometric analysis.
References
- Huang, F.L. and Glinsmann, W.H. (1976) Eur. J. Biochem., 70, 419-426.
- Foulkes, J.G. and Cohen, P. (1980) Eur. J. Biochem., 105, 195-203.
- Park, I., Roach, P., Bonder, J., Fox, S.P. and DePaoli-Roach, A.A. (1994) J. Biol. Chem., 269, 944-954.
- Ingebritsen, T.S. and Cohen, P. (1983) Eur. J. Biochem., 132, 255-261.
- Cohen, P. (1991) Methods Enzymol., 201, 389-398.
Reagents Sold Separately
PP1 Reaction Buffer Pack
Companion Products
Protein Phosphatase 1 (PP1)
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