Recombinant Human Active Chk1 Protein, CF

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The approximate molecular weight is 82 kDa and the average purity is 95%.

Product Details

Summary
Reactivity HuSpecies Glossary
Applications Bioactivity
Format
Carrier-Free

Order Details

Recombinant Human Active Chk1 Protein, CF Summary

Details of Functionality
The activity of Chk1 is typically 169-229 nmol/min/mg using a synthetic peptide substrate (KKKVSRSGLYRSPSMPENLNRPR) (see Activity Assay Protocol).
Source
Spodoptera frugiperda, Sf 9 (baculovirus)-derived human Chk1 protein
Accession # NM_001274
Accession #
N-terminal Sequence
Using an N-terminal GST tag
Protein/Peptide Type
Recombinant Enzymes
Gene
CHEK1
Purity
>70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane

Applications/Dilutions

SDS-PAGE
82 kDa
Publications
Read Publication using
1630-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, 0.25 mM DTT, 0.1 mM EGTA, 0.1 mM EDTA, 0.1 mM PMSF, 25% glycerol.
Purity
>70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Assay Procedure
  • Active Kinase - Active Chk1 (0.1 μg/μL) diluted with Kinase Dilution Buffer to the concentrations shown 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 Chk1, 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 Chk1: 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)]

Notes

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

Alternate Names for Recombinant Human Active Chk1 Protein, CF

  • Checkpoint, S. pombe, homolog of, 1
  • CHEK1
  • CHK1 (checkpoint, S.pombe) homolog
  • CHK1 checkpoint homolog (S. pombe)
  • Chk1
  • CHK1serine/threonine-protein kinase Chk1
  • EC 2.7.11
  • EC 2.7.11.1

Background

Chk1 is a 56 kDa Serine/Threonine protein kinase that was originally identified in fission yeast to play a role in activation of the DNA damage checkpoint in the G2 phase of the cell cycle (1). Chk1 appears to function downstream of several of the known fission yeast checkpoint gene products, including that encoded by Rad3, a gene with sequence similarity to the ATM gene mutated in patients with ataxia telangiectasia (2).
  1. Walworth, N. et al. (1993) Nature 363:368.
  2. Walworth, N.C. et al. (1996) Science 271:353.

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Publications for Chk1 (1630-KS)(1)

We have publications tested in 1 confirmed species: N/A.

We have publications tested in 1 application: Bioassay.


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Bioinformatics

Gene Symbol CHEK1
Uniprot