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  • p42 MAP Kinase (MAPK)

    Description

    p42 MAP Kinase (Mitogen-Activated Protein Kinase, MAPK), also known as Erk2 (Extracellular signal-regulated kinase 2) is one of two isoforms of MAP kinase family. It is a serine/threonine protein kinase, participating in mammalian signal transduction pathways that control intracellular responses to hormones and major developmental changes. Full activation of p42 MAP Kinase requires phosphorylation at residues T183 and Y185 catalyzed by the upstream protein kinase MEK2 or MAP Kinase Kinase (MAPKK) (1-4). 

    Highlights

    • Protein serine/threonine kinase
    • Mouse, recombinant (E. coli)

    Product Source

    Isolated from a strain of E. coli that carries a clone expressing murine MAPK (1) under the control of a T7 expression system. Fully active MAPK is produced by co-expression with a constitutively active form of its activator, MEK2 (6).

    Recognition Determinant

    The minimal recognition motif for phosphorylation by MAPK is S/TP. Pro is also common at the -2 position in the optimal primary motif PXS/TP. The substrate specificity of MAPK overlaps with other proline-directed protein kinases present within the cell. This suggests that the recognition of protein substrates may require structural determinants in addition to primary sequence requirements (5).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer for Protein Kinases (PK)-2010X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of MAPK required to catalyze the transfer of 1 pmol of phosphate from ATP to myelin basic protein (50 µM) in 1 minute at 30°C in a total reaction volume of 30 µl.

    Reaction Conditions

    1X NEBuffer for Protein Kinases (PK)
    Incubate at 30°C

    1X NEBuffer for Protein Kinases (PK):
    50 mM Tris-HCl
    10 mM MgCl2
    0.1 mM EDTA
    2 mM DTT
    0.01% Brij 35
    pH 7.5 @ 25°C

    Storage Temperature

    -70°C

    Storage Conditions

    50 mM HEPES
    100 mM NaCl
    1 mM DTT
    50% Glycerol
    0.1 mM EDTA
    0.01% Brij 35
    pH 7.5 @ 25°C

    Heat Inactivation

    65°C for 20 min

    Molecular Weight

    Theoretical: 42 kDa

    Specific Activity

    10,000,000 units/mg

    Storage Notes

    • Avoid repeated freeze/thaw cycles.

    Shipping Notes

    • Ships on dry ice

    Quality Control

    Quality Assurance Statement

    • p42 MAP Kinase (Erk2) contains no detectable protease, phosphatase or MEK Kinase activities.

    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.
    • MEK Kinase Activity:

      The Kinase is tested in a reaction with an unphosphorylated MAP Kinase as a substrate. After incubation, there is no detectable MEK Kinase activity as determined by phosphocellulose paper binding assay.

    • Phosphatase Activity (PNPP):
      The product is tested in a reaction containing a p-nitrophenyl phosphate (PNPP), a chromogenic substrate for most phosphatases. After incubation there is no detectable phosphatase activity as determined by spectrophotometric analysis of released p-nitrophenol at 405 nm.
    • 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. General notes:
      •  p42 MAP Kinase has been purified to >95% homogeneity as determined by SDS-PAGE and Coomassie Blue staining. 
      • Avoid freeze/thaw cycles. Can be stored for 2 weeks or less at -20°C. 
      • If the source of protein to be phosphorylated is a crude extract of cells or tissue, it is very important to include the appropriate protease and protein phosphatase inhibitors in the lysis buffer and to use shorter incubation time for phosphorylation. 
      • Reaction Conditions: 1X p42 MAP Kinase Reaction Buffer, supplemented with 200 µM ATP and gamma phosphate-labeled ATP to a final specific activity of 100-500 µCi/µmol.
    2. Usage notes:
      • Optimal incubation times and enzyme concentrations must be determined empirically for each particular substrate. 
      • If possible, the ATP concentration should be at or near saturation (5 -10-fold over Km). Apparent Km values of ATP for most protein kinases are below 100 μM. 
      However, if the objective is to measure enzyme activity using gamma phosphate-labeled ATP, it is best to use 100-200 μM ATP in order to have higher specific activity of gamma-labeled ATP (100-500 cpm/pmol). Also, an excess of substrate should be used, and the level of phosphorylation should not exceed 10% for determination of the initial rate. 

      To phosphorylate a protein or peptide substrate to completion, the ATP concentration should be about 5-fold over the limited substrate concentration. Higher enzyme concentration and prolonged incubation times should be employed. 

      Recommended reference: Protein Phosphorylation: A Practical Approach (1993) ed. Hardie, D.G. IRL Press.

    References

    1. Boulton, T.G. Cell. 65
    2. Rossomando, A.J. et al. J. Biol. Chem.. 266
    3. Payne, D.M. et al. EMBO J.. 10
    4. Prowse, C.N. et al. J. Biol. Chem.. 276
    5. Davis, R.J. J. Biol. Chem.. 268
    6. Wu, J. et al. (1993). Mol. Cell Biol.. 13, 4539-4548.

    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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