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).
Recombinant Human Active Chk2 Protein, CF
R&D Systems | Catalog # 1358-KS
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Key Product Details
- R&D Systems Sf 9 (baculovirus)-derived Recombinant Human Active Chk2 Protein (1358-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
Applications
Bioactivity
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Product Specifications
Source
Spodoptera frugiperda, Sf 9 (baculovirus)-derived human Chk2 protein
Purity
>90%, 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
88 kDa
Activity
The specific activity of Chk2 was determined to be 660 nmol/min/mg using a synthetic peptide substrate (KKKVSRSGLYRSPSMPENLNRPR).
Scientific Data Images for Recombinant Human Active Chk2 Protein, CF
Recombinant Human Active Chk2 Protein SDS-PAGE.
The approximate molecular weight is 88 kDa and the purity is > 90%.Formulation, Preparation, and Storage
1358-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: Chk2
References
- Matsuoka, S. et al. (1998) Science 282:1893.
- Chehab, N.H. et al. (2000) Genes Dev. 14:278.
Long Name
Checkpoint Kinase 2
Alternate Names
CDS1, CHEK2, HuCds1, LFS2, PP1425, Rad53
Gene Symbol
CHEK2
UniProt
Additional Chk2 Products
Product Documents for Recombinant Human Active Chk2 Protein, CF
Certificate of Analysis
To download a Certificate of Analysis, please enter a lot or batch number in the search box below.
Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Recombinant Human Active Chk2 Protein, CF
For research use only
Citations for Recombinant Human Active Chk2 Protein, CF
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Protocols
View specific protocols for Recombinant Human Active Chk2 Protein, CF (1358-KS):
Materials
- 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.
- Thaw the [33P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area.
- Thaw the Active Chk2, Kinase Assay Buffer, Substrate, and Kinase Dilution Buffer on ice.
- 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 - 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.
- 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.
- 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.
- 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.
- Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
- 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)]