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  • Lambda Protein Phosphatase (Lambda PP)

    Description

    Lambda Protein Phosphatase (Lambda PP) is a Mn2+-dependent protein phosphatase with activity towards phosphorylated serine, threonine and tyrosine residues. It is the 221 amino-acid product of the ORF221 open reading frame on bacteriophage lambda (1,2).


    Highlights

  • Dual Specificity: protein serine/threonine/tyrosine phosphatase 
  • Bacteriophage lambda, recombinant (E. coli)
  • Product Source

    Isolated from a strain of E. coli that carries the bacteriophage lambda ORF221 open reading frame under the control of a T7 expression system (kindly provided by Dr. D. Barford) (2).

    Reagents Supplied

    The following reagents are supplied with this product:

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

    Advantages and Features

    Applications

    • Lambda PP 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
    100 mM NaCl
    2 mM DTT
    0.01% Brij 35
    0.1 mM EGTA
    0.1 mM MnCl2
    50% Glycerol
    pH 7.5 @ 25°C

    Heat Inactivation

    65°C for 60 min

    Molecular Weight

    Theoretical: 25 kDa

    Specific Activity

    800,000 units/mg

    Storage Notes

    • Avoid repeated freeze/thaw cycles.

    Shipping Notes

    • Ships on dry ice

    Quality Control

    Quality Assurance Statement

    • Lambda PP has been purified to >95% homogeneity as determined by SDS-PAGE and Coomassie Blue staining.

    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 Lambda PP.
    2. 100 units of Lambda PP remove ~100% of phosphates (0.5 nmol) in phosphorylated myelin basic protein (phospho-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 Lambda PP is assessed on MyBP phosphorylated exclusively on serine/threonine residues with cAMP-dependent Protein Kinase. The Protein Tyrosine Phosphatase (PTP) activity is assessed on MyBP phosphorylated exclusively on tyrosine residues with Abl Protein Tyrosine Kinase.
    4. Lambda PP is active on phosphorylated histidine residues (2).
    5. 0ptimal incubation times and enzyme concentrations must be determined empirically for each particular substrate. 
    6. 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.
    7. The following levels of inhibition of Lambda PP (100 units) are found when the reaction buffer and MnCl2 are supplemented with: 
      • 10 mM Sodium Orthovanadate (3): 80% 
      • 10 mM Sodium Orthovanadate, 50 mM Sodium Fluoride: 90%
      • 50 mM EDTA: 95% 
      • 1% Triton X-100: no 
      • 0.4% Nonidet P-40: no 
      • 0.025% Tween 20: no 
      • 0.5 M NaCl: 5% 
      • ATP Mix (10 mM MgCl2, 0.1 mM ATP): no
      • Protease Inhibitor Cocktail*: 10% 
    8. *Pepstatin A, leupeptin and aprotinin, 10 µg/ml each, 0.5 mM PMSF and 1 mM benzamidine

    References

    1. Cohen, P.T.W. and Cohen, P. (1989). Biochem. J.. 260, 931-934.
    2. Zhuo, S. et al. (1993). J. Biol. Chem.. 268, 17754-17761.
    3. 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.
    1. Can I use the enzyme to de-phosphorylate proteins?

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