Recombinant Human Active Chk2 Protein, CF

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  • Purity
    >70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
  • Activity
    The activity of Chk2 is typically 654-884 nmol/min/mg using a synthetic peptide substrate (KKKVSRSGLYRSPSMPENLNRPR) (see Activity Assay Protocol).
  • Source
    Spodoptera frugiperda, Sf 9 (baculovirus)-derived Accession # NM_007194
  • Accession #
  • N-terminal Sequence

    Using an N terminal GST tag

    88 kDa
Formulation Supplied in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.25 mM DTT, 0.1 mM EGTA, 0.1 mM EDTA, 0.1 mM PMSF, 25% glycerol.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage: This product is stable at ≤ ‑70° C for up to 1 year from the date of receipt. For optimal storage, aliquot into smaller quantities after centrifugation and store at recommended temperature. Avoid repeated freeze-thaw cycles.
Assay Procedure
  • Active Kinase - Active Chk2 (0.1 μg/μL) diluted with Kinase Dilution Buffer and assayed as outlined in Figure 2. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
  • Kinase Assay Buffer I, pH 7.2 - 25 mM MOPS, 12.5 mM beta -glycerolphosphate, 25 mM MgCl2, 5 mM EGTA, 2 mM EDTA. Add 0.25 mM DTT to the Kinase Assay Buffer prior to use.
  • Kinase Dilution Buffer, pH 7.2 - Kinase Assay Buffer I diluted at a 1:4 ratio (5-fold dilution) with distilled or deionized water.
  • 10 mM ATP Stock Solution - Prepare the ATP Stock Solution by dissolving 55 mg of ATP in 10 mL of Kinase Assay Buffer I.
  • [33P]-ATP Assay Cocktail - Prepare 250 μM [33P]-ATP Assay Cocktail in a designated radioactive work area by combining 150 μL of 10 mM ATP Stock Solution, 100 μL of [33P]-ATP (1 mCi/100 μL), and 5.75 mL of Kinase Assay Buffer I.
  • Substrate - Chk-tide synthetic peptide substrate (KKKVSRSGLRSPSMPENLNRPR) diluted in distilled or deionized water to a final concentration of 1 mg/mL.
  1. Thaw the [33P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area.
  2. Thaw the Active Chk2, Kinase Assay Buffer I, Substrate, and Kinase Dilution Buffer on ice.
  3. In a pre-cooled microfuge tube, add the following reaction components bringing the initial reaction volume up to 20 μL.
    a. Diluted Active Chk2: 10 μL
    b. Substrate (1 mg/mL Stock Solution): 5 μL
  4. Set up the blank control as outlined in step 3, excluding the addition of the substrate. Replace the substrate with an equal volume of distilled or deionized water.
  5. Initiate the reaction by the addition of 5 μL [33P]-ATP Assay Cocktail, bringing the final volume up to 25 μL. Incubate the mixture in a water bath at 30 °C for 15 minutes.
  6. After the 15 minute incubation period, terminate the reaction by spotting 20 μL of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper.
  7. Air dry the pre-cut P81 strip and sequentially wash in a 1% phosphoric acid solution (dilute 10 mL of phosphoric acid and make a 1 liter solution with distilled or deionized water) with constant gentle stirring. It is recommended that the strips be washed a total of three times for approximately 10 minutes each.
  8. Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
  9. Determine the corrected cpm by removing the blank control value (see step 4) for each sample and calculate the kinase specific activity as outlined below:

    Calculation of [33P]-ATP Specific Activity (SA) (cpm/pmol)
    Specific Activity (SA) = cpm for 5 μL [33P]-ATP/pmol of ATP (in 5 μL of a 250 μM ATP stock solution, i.e. 1250 pmol)

    Calculation of Kinase Specific Activity (SA) (pmol/minutes/μg or nmol/minutes/mg)
    Corrected cpm from reaction / [(SA of 33P-ATP in cpm/pmol) x (Reaction time in minutes) x (Enzyme amount in μg or mg)] x [(Reaction volume) / (Spot Volume)]
Data Images
The approximate molecular weight is 88 kDa and the average purity is 90%.
Background: Chk2

Chk2 is rapidly phosphorylated and activated in response to replication blocks and DNA damage where the response to DNA damage occurs in an ataxia telangiectasia mutated (ATM)-dependent manner (1). Expression of wild-type Chk2 leads to increased p53 stabilization after DNA damage, whereas expression of a dominant-negative Chk2 mutant abrogates both phosphorylation of p53 on Serine 20 and p53 stabilization (2).

  • References:
    1. Matsuoka, S. et al. (1998) Science 282:1893.
    2. Chehab, N.H. et al. (2000) Genes Dev. 14:278.
  • Long Name:
    Checkpoint Kinase 2
  • Entrez Gene IDs:
    11200 (Human); 50883 (Mouse); 114212 (Rat)
  • Alternate Names:
    CDS1; CHEK2; CHK2 checkpoint homolog (S. pombe); EC 2.7.11; EC; HuCds1; LFS2; PP1425; Rad53; S.pombe) homolog
Related Research Areas

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

Showing Results 1 - 1 of 1

  1. Chk2 phosphorylation of survivin-DeltaEx3 contributes to a DNA damage-sensing checkpoint in cancer.
    Authors: Lopergolo A, Tavecchio M, Lisanti S, Ghosh J, Dohi T, Faversani A, Vaira V, Bosari S, Tanigawa N, Delia D, Kossenkov A, Showe L, Altieri D
    Cancer Res, 2012;72(13):3251-9.
    Species: Human
    Sample Type: recombinant survivin deltaEx3
    Application: Kinase Assay

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