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Recombinant Human Active Chk2 Protein, CF

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The approximate molecular weight is 88 kDa and the average purity is 90%.
The approximate molecular weight is 88 kDa and the average purity is 90%.

Product Details

Summary
Reactivity HuSpecies Glossary
Applications Bioactivity
Format
Carrier-Free

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Recombinant Human Active Chk2 Protein, CF Summary

Details of Functionality
The specific activity of Chk2 was determined to be 660 nmol/min/mg using a synthetic peptide substrate (KKKVSRSGLYRSPSMPENLNRPR).
Source
Spodoptera frugiperda, Sf 9 (baculovirus)-derived human Chk2 protein
Accession #
N-terminal Sequence

Using an N terminal GST tag
Protein/Peptide Type
Recombinant Enzymes
Gene
CHEK2
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Applications/Dilutions

Dilutions
  • Bioactivity
SDS-PAGE
88 kDa
Publications
Read Publication using
1358-KS in the following applications:

Packaging, Storage & Formulations

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.
Buffer
Supplied in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM glutathione, 0.1 mM EDTA, 0.25 mM DTT, 0.1 mM PMSF, 25% glycerol.
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
Assay Procedure
  • Active Kinase - Active Chk2 (0.1 μg/μL) diluted with Kinase Dilution Buffer and assayed as outlined in Sample Activity Plot. Note: These are suggested working dilutions and it is recommended that the researcher perform a serial dilution of Active Chk2 for optimal results..
  • Kinase Assay Buffer - 25 mM MOPS, pH 7.2, 12.5 mM beta -glycerol-phosphate, 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 - Kinase Assay Buffer diluted at a 1:4 ratio (5X dilution) with distilled water.
  • 10 mM ATP Stock Solution - Prepare the ATP Stock Solution by dissolving 55 mg of ATP in 10 mL of Kinase Assay Buffer. Store 200 μL aliquots at -20 °C.
  • [33P]-ATP Assay Cocktail - Prepare 250 μM [33P]-ATP Assay Cocktail in a designated radioactive work area by adding the following components: 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. Store 1 mL aliquots at -20 °C.
  • Substrate - Chktide synthetic peptide substrate (KKKVSRSGLRSPSMPENLNRPR) diluted in distilled 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, 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. Stock Solution of Substrate (1 mg/mL ): 5 μL
    c. Distilled water (2-8 °C): 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 water.
  5. Initiate the reaction by the addition of 5 μL [33P]-ATP Assay Cocktail, bringing the final volume up to 25 μL and 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 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)]

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Active Chk2 Protein, CF

  • CDS1
  • CHEK2
  • CHK2 checkpoint homolog (S. pombe)
  • Chk2
  • EC 2.7.11
  • EC 2.7.11.1
  • HuCds1
  • LFS2
  • PP1425
  • Rad53
  • S.pombe) homolog

Background

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).

  1. Matsuoka, S. et al. (1998) Science 282:1893.
  2. Chehab, N.H. et al. (2000) Genes Dev. 14:278.

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Publications for Chk2 (1358-KS)(1)

We have publications tested in 1 confirmed species: Human.

We have publications tested in 1 application: Bioassay.


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Bioinformatics

Gene Symbol CHEK2
Uniprot