Recombinant Human Active Chk1 Protein, CF

R&D Systems | Catalog # 1630-KS

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Key Product Details

  • R&D Systems Sf 9 (baculovirus)-derived Recombinant Human Active Chk1 Protein (1630-KS)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

Sf 9 (baculovirus)

Accession Number

NM_001274

Applications

Bioactivity
View all Chk1 Proteins and Enzymes »

Product Specifications

Source

Spodoptera frugiperda, Sf 9 (baculovirus)-derived human Chk1 protein

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

N-terminal Sequence Analysis

Using an N-terminal GST tag

SDS-PAGE

82 kDa

Activity

The specific activity of Chk1 was determined to be 228 nmol/min/mg using a synthetic peptide substrate (KKKVSRSGLYRSPSMPENLNRPR).

Scientific Data Images for Recombinant Human Active Chk1 Protein, CF

Recombinant Human Active Chk1 Protein SDS-PAGE

Recombinant Human Active Chk1 Protein SDS-PAGE.

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

Formulation, Preparation, and Storage

1630-KS
Formulation 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.
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.

Background: Chk1

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).

References

  1. Walworth, N. et al. (1993) Nature 363:368.
  2. Walworth, N.C. et al. (1996) Science 271:353.

Long Name

Checkpoint Kinase 1

Alternate Names

CHEK1

Entrez Gene IDs

1111 (Human); 12649 (Mouse); 140583 (Rat)

Gene Symbol

CHEK1

UniProt

NM_001274 (Human)

Additional Chk1 Products

Product Documents for Recombinant Human Active Chk1 Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

Product Specific Notices for Recombinant Human Active Chk1 Protein, CF

For research use only

Citations for Recombinant Human Active Chk1 Protein, CF

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Protocols

View specific protocols for Recombinant Human Active Chk1 Protein, CF (1630-KS):

Materials
  • Active Kinase - Active Chk1 (0.1 μg/μL) diluted with Kinase Dilution Buffer I 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 - 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 I prior to use.
  • Kinase Dilution Buffer - Kinase Assay Buffer I diluted at a 1:4 ratio (5-fold) 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 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. Store 1.0 mL aliquots at ≤ -20 °C.
  • 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, 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): 5.0 μL
    c. Distilled water: 5.0 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. Incubate the mixture in a water bath at 30 °C for 15 minutes.
  6. After the 15 minute incubation, 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 (add 10 mL of phosphoric acid to 990 mL of distilled 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)]

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