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Protein Phosphatase 1 (PP1) |
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- Protein serine/threonine phosphatase
- Rabbit skeletal muscle, recombinant (E. coli)
- Supplied with 10X Reaction Buffer
Description:
Protein Phosphatase 1 (PP1) is a Mn2+-dependent protein phosphatase with activity towards phosphoserine/threonine residues. It consists of the 330 amino-acid catalytic subunit of the α-isoform of type 1 protein phosphatase from rabbit skeletal muscle (1,2). Recombinant PPI shows some activity towards phosphotyrosine residues (3,4).
Source:
Isolated from a strain of E.coli that carries the coding sequence for rabbit skeletal muscle PP1 under the control of the trp-lac hybrid promoter (kindly provided by Dr. E.Y.C. Lee) (1,2).
Applications:- PP1 can be used to release phosphate groups from phosphorylated serine, threonine and tyrosine residues in proteins. Note that different proteins are dephosphorylated at different rates.
Reagents Supplied:
PP1 Reaction Buffer Pack (10X)
MnCl2 (10X)
Enzyme Properties
Heat Inactivation:
65°C for 1 hour
Molecular Weight:
Theoretical: 37.5 kDa
Specific Activity:
25,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X PP1 Reaction Buffer
Supplemented with 1 mM MnCl2
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 enzyme that hydrolyzes 1 nmol of p-nitrophenyl phosphate (50 mM) in 1 minute at 30°C in a total reaction volume of 50 μl.
Concentration:
2,500 units/ml
Storage Conditions:
50 mM HEPES
200 mM NaCl
1 mM MnCl2
2.5 mM Dithiothreitol
0.1 mM EGTA
50% Glycerol
0.025% Tween-20
pH 7.0 @ 25°C
Storage Temperature:
-70°C
Avoid repeated freeze/thaw cycles.
Notes
General notes:- PP1 has been purified to > 90% homogeneity as determined by SDS-PAGE and Coomassie Blue staining.
- Avoid freeze/thaw cycles. Can be stored for 1 week or less at -20°C.
Usage notes:- The following information can be used as suggested initial conditions for dephosphorylation of proteins with PP1.
0.1 unit of PP1 removes ~100% of phosphates (0.5 nMol) from phosphoserine/threonine residues in phosphorylase a as well as in myelin basic protein (MyBP, 18.5 kDa) in 30 minutes in a 50 µl reaction. The concentration of phosphorylated protein is 10 µM with respect to phosphate.
The Protein Serine/threonine Phosphatase (PSP) activity of PP1 is assessed on phosphorylase a phosphorylated on a serine residue with phosphorylase kinase, and also on MyBP phosphorylated on serine/threonine residues with cAMP-dependent Protein Kinase using the PSP Assay System (NEB #P0780S).
Note that optimal incubation times and enzyme concentrations must be determined empirically for each particular substrate.
It has been found that bacterially expressed catalytic subunits of the α- and γ- isoforms of type 1 protein phosphatase can dephosphorylate phosphotyrosine residues in proteins (3,4). We assessed the Protein Tyrosine Phosphatase (PTP) activity of PP1 on MyBP phosphorylated on tyrosine residues with Abl Protein Tyrosine Kinase using the PTP Assay System (NEB #P0785S). We have measured the PTP activity as high as 30% of the PSP activity measured on MyBP phosphorylated on serine/threonine residues.
PP1 has been shown to be active on phosphorylated histidine residues (5).
The following levels of inhibition of PP1 (0.1 unit) are found when the reaction buffer is supplemented with:
1 µM Protein Phosphatase
Inhibitor 2 (NEB #P0755): 100%
10 µM okadaic acid: 85%
0.1 µM microcystin-LR: 100%
10 mM sodium orthovanadate (6): 95%
50 mM sodium fluoride: 40%
50 mM Na2 EDTA: 95%
1% Triton X-10: 5%
0.4% Nonidet P-40: no inhibition
0.5 M NaCl: no inhibition
Protease Inhibitor Cocktail*: no inhibition
*Pepstatin A, leupeptin and aprotinin, 10 µg/ml each, 0.5 mM PMSF, and 1 mM benzamidine
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:
PP1 contains no detectable protease activity.
Protease Activity:
After incubation of 50 units of Protein Phosphatase 1 (PP1) with a standard mixture of proteins
for 2 hours at 30ºC, no proteolytic activity could be detected by SDS-PAGE.
References
- Bai, G., Zhang, Z., Amin, J., Deans-Zirattu, S.A. and Lee, E.Y.C. (1988) FASEB J., 2, 3010-3016.
- Zhang, Z., Bai, G., Deans-Zirattu, S., Browner, M.F. and Lee, E.Y.C. (1992) J. Biol. Chem., 267, 1484-1490.
- Barshevsky, T. and Roberts, R.J. (1997) The NEB Transcript, 8(2), 14.
- MacKintosh, C., Garton, A.J., McDonnell, A., Barford, D., Cohen, P.T.W., Tonks, N.K. and Cohen, P. (1996) FEBS Lett., 397, 225-238.
- Kim, Y., Huang, J., Cohen, P. and Matthews, H.R. (1993) J. Biol. Chem., 268, 18513-18518.
- Gordon, J.A. (1991) Methods Enzymol., 201, 477-482.
Reagents Sold Separately
PP1 Reaction Buffer Pack
Companion Products
Protein Phosphatase Inhibitor 2 (I-2)
Protein Ser/Thr Phosphatase (PSP) Assay System
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