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  • Protein Phosphatase 1 (PP1)

    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).


    Highlights

  • Protein serine/threonine phosphatase
  • Rabbit skeletal muscle, recombinant (E. coli)
  • Product 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).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer for PMP-2010X
    MnCl210X

    Advantages and Features

    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.

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that hydrolyzes 1 nmol of p-Nitrophenyl Phosphate (50 mM) (NEB #P0757) in 1 minute at 30°C in a total reaction volume of 50 μl.

    Reaction Conditions

    1X NEBuffer for PMP
    Supplement with 1 mM MnCl2
    Incubate at 30°C

    1X NEBuffer Pack for Protein MetalloPhosphatases (PMP):
    50 mM HEPES
    10 mM NaCl
    2 mM DTT
    0.01% Brij 35
    pH 7.5 @ 25°C

    Storage Temperature

    -70°C

    Storage Conditions

    50 mM HEPES
    200 mM NaCl
    2.5 mM DTT
    50% Glycerol
    0.02% Tween® 20
    0.1 mM EGTA
    pH 7.0 @ 25°C

    Heat Inactivation

    65°C for 60 min

    Molecular Weight

    Theoretical: 37.5 kDa

    Specific Activity

    80,000 units/mg

    Storage Notes

    • Avoid repeated freeze/thaw cycles.
    • Can be stored for one week of less at -20°.

    Shipping Notes

    • Ships on dry ice

    Quality Control

    Quality Assurance Statement

    • PP1 contains no detectable protease activity.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Protease Activity (SDS-PAGE):

      The product is tested for protease activity by incubation with a standard mixture of proteins resulting in no detectable degradation of the proteins as determined by SDS-PAGE.

    Notes

    1. The following information can be used as suggested initial conditions for dephosphorylation of proteins with PP1.
    2. 0.1 unit of PP1 removes ~100% of phosphates (0.5 nmol) from phosphoserine/threonine residues in phosphorylase a as well as in phosphorylated myelin basic protein (MyBP, 18.5 kDa) in 30 minutes in a 50 µl reaction. The concentration of phospho-MyBP is 10 µM with respect to phosphate.
    3. 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. 
    4. Optimal incubation times and enzyme concentrations must be determined empirically for each particular substrate.
    5. 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. We have measured the PTP activity as high as 10-30% of the PSP activity measured on MyBP phosphorylated on serine/threonine residues.
    6. PP1 has been shown to be active on phosphorylated histidine residues (5). 
    7. If the source of phosphorylated protein is a crude extract of cells or tissue, it is very important to include the appropriate protease inhibitors in the lysis buffer and to use shorter incubation time for dephosphorylation.
    8. 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) (NEB #P0758): 95%
      • 50 mM Sodium Fluoride (NEB #P0759): 40%
      • 50 mM 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

    References

    1. Bai, G., Zhang, Z., Amin, J., Deans-Zirattu, S.A. and Lee, E.Y.C. (1988). FASEB J.. 2, 3010-3016.
    2. Zhang, Z., Bai, G., Deans-Zirattu, S., Browner, M.F. and Lee, E.Y.C. (1992). J. Biol. Chem.. 267, 1484-1490.
    3. Barshevsky, T. and Roberts, R.J. (1997). The NEB Transcript. 8(2)
    4. 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.
    5. Kim, Y., Huang, J., Cohen, P. and Matthews, H.R. (1993). J. Biol. Chem.. 268, 18513-18518.
    6. Gordon, J.A. (1991). Methods Enzymol.. 201, 477-482.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

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