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, and 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.
Active Kinase - Active COT (0.1 μg/μL) diluted with Kinase Dilution Buffer. 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 5-fold with a 50 ng/μL BSA solution.
10 mM ATP Stock Solution - Prepare the ATP Stock Solution by dissolving 55 mg of ATP in 10 mL of Kinase Assay Buffer I. 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 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 mL aliquots at ≤ -20 °C.
Substrate - Inactive MEK1 and ERK1 were activated in a coupled reaction. Myelin Basic Protein (MBP) diluted in distilled or deionized water to a final concentration of 1 mg/mL was subsequently used as a substrate for the activated ERK1.
Thaw the Active COT, Kinase Assay Buffer I, and inactive MEK1 and ERK1 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 COT: 10 μL b. Inactive MEK1 (0.2 μg/μL): 2 μL c. Inactive ERK1 (0.2 μg/μL): 3 μL d. Kinase Dilution Buffer: 5 μL
Start the reaction with the addition of 5 μL ATP (250 μM) and incubate in a water bath at 30 °C for 25 minutes.
After the 25 minute incubation, remove 5 μL and add it to the following reaction components on ice, bringing the initial reaction volume up to 20 μL. a. Reaction Mixture: 5 μL b. Distilled or deionized water (on ice): 10 μL c. MBP Substrate (1.0 mg/mL; on ice): 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 or deionized water.
Thaw the [33P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area. Initiate the reaction with 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.
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.
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 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.
Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
Determine the corrected cpm by subtracting 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/pmole 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)]
The approximate molecular weight is 70 kDa and the average purity is 75%.
COT is an oncogene that can activate both the MAP kinase and JNK kinase pathways. COT activates I kappa B kinases and induces the nuclear production of NF-kappa B. The C-terminal catalytic domain of KSR2 associates with COT and KSR2 can negatively regulate the kinase activity of COT in vitro. Co-transfection of KSR2 with COT in cells lead to reduced COT-mediated ERK activation and COT induced IL-8 production in a dose-dependent manner (1). COT is one of the MAP kinase kinase (MAPKK) kinases that regulates the ERK1/ERK2 pathway in response to IL-1. Blockage of expression of COT results in failure of IL-1 to induce an increase in IL 8 and MIP-1 beta mRNA levels (2).
Channavajhala, P.L. et al. (2003) J. Biol. Chem. 278:47089.
Rodriguez, C. et al. (2006) Cell Signal. 18:1376.
Mitogen-activated Protein Kinase Kinase Kinase 8
Entrez Gene IDs:
1326 (Human); 26410 (Mouse); 116596 (Rat)
Cancer Osaka thyroid oncogene; c-COT; cot (cancer Osaka thyroid) oncogene; COT; COTMEKK8; EC 126.96.36.199; EST; ESTF; ESTFLJ10486; Ewing sarcoma transformant; MAP3K8; mitogen-activated protein kinase kinase kinase 8; Proto-oncogene c-Cot; proto-oncogene serine/threoine protein kinase; Serine/threonine-protein kinase cot; TPL2; Tpl-2; Tumor progression locus 2; tumor progression locus-2
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