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  • Abl Protein Tyrosine Kinase (Abl)

    Discontinued Date

    01/01/2013
    recombinant unique buffer heat inactivation
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    Discontinued Products

    Description

    Abl Protein Tyrosine Kinase (AbI) is a truncated form of the v-AbI Protein Tyrosine Kinase, a partner in the Gag-Abl fusion protein of the Abelson murine leukemia virus. Abl contains 407 amino acids (residues 237-643 of the p120-gag-abl polyprotein), which include the kinase catalytic domain, SH2 domain on the N-terminus and the I237M mutation. This truncated form of v-Abl is identical to the normal c-Abl (1-3). Abl is autophosphorylated on tyrosine(s)(2). 

    Highlights

    • Protein tyrosine kinase
    • Abelson Murine Leukemia Virus, recombinant (E. coli)

    Product Source

    Isolated from a strain of E. coli that carries the truncated AbI Protein Kinase encoded by the Abelson murine leukemia virus under the control of a T7 expression system (kindly provided by Dr. S. Goff).

    Recognition Determinant

    The recognition motif for phosphorylation by Abl is I/V/LYXXP/F. Abl, like many cytosolic protein tyrosine kinases, preferentially phosphorylates sites recognized by its own SH2 domain, selects substrates with large hydrophobic amino acids at the +3 position and β-branched amino acids at the -1 position (4).

    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 Abl required to catalyze the transfer of 1 pmol of phosphate to the Abl Peptide Substrate, EAIYAAPFAKKK (100 µM) in 1 minute at 30°C in a total reaction volume of 25 µl (4).

    Supplemented with 200 μM ATP and 300 μCi/μmol gamma-labeled ATP


    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

    Usage Concentration

    100,000 units/ml

    Storage Temperature

    -70°C

    Storage Conditions

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

    Heat Inactivation

    65°C for 20 min

    Molecular Weight

    Theoretical: 45 kDa

    Specific Activity

    13,000,000 units/mg

    Shipping Notes

    • Ships on dry ice

    Quality Control

    Quality Assurance Statement

    • Abl contains no detectable protease or phosphatase 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.
    • 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. Optimal incubation times and enzyme concentrations must be determined empirically for each particular substrate.
    2. Abl Protein Kinase phosphorylates the Abl Peptide Substrate with a Km of 4 μM(4).
    3. The recombinant Abl is autophosphorylated at tyrosine residues when expressed in E. coli. The autophosphorylation was confirmed by Western blot analysis using the phospho-tyrosine specific antibody.
    4. 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-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.
    5. Avoid freeze/thaw cycles. Can be stored for 2 weeks or less at -20°C.
    6. 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.
    7. Reaction Conditions for Unit Definition Assay: 1X supplemented with 200 µM ATP and gamma-labeled ATP to a final specific activity of 100-500 µCi/µmol.

    References

    1. Reddy, P.E. et al. (1983). Proc. Natl. Acad. Sci. USA. 80, 3623-3627.
    2. Foulkes, J.G. et al. (1985). J. Biol. Chem.. 260, 8070-8077.
    3. Oppi, C. et al. (1987). Proc. Natl. Acad. Sci. USA. 84, 8200-8204.
    4. Songyang, Z. et al. (1995). Nature. 373, 536-539.

    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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