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 MEK2 (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.
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.
[32P]-ATP Assay Cocktail - Prepare 250 μM [32P]-ATP Assay Cocktail in a designated radioactive work area by combining 150 μL of 10 mM ATP Stock Solution, 100 μL of [32P]-ATP (1 mCi/100 μL), and 5.75 mL of Kinase Assay Buffer. Store 1 mL aliquots at ≤ -20 °C.
Substrate - Inactive ERK2 was activated using MEK2 and a Myelin Basic Protein (MBP) substrate was diluted in distilled or deionized water to a final concentration of 1 mg/mL.
Thaw the Active MEK2, Kinase Assay Buffer I, and inactive ERK2 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 MEK2: 5 μL b. Inactive ERK2 (0.2 μg/μL): 10 μL c. Kinase Assay 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 15 minutes.
After the 15 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. MBP Substrate (1 mg/mL; on ice): 5 μL c. Distilled or deionized water (on ice): 10 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 [32P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area. Initiate the reaction with the addition of 5 μL [32P]-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 [32P]-ATP Specific Activity (SA) (cpm/pmol) Specific Activity (SA) = cpm for 5 μL [32P]-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 32P-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 71 kDa and the average purity is 90%.
MEK2 is the member of MAPK kinase (MAPKK) family of signaling protein kinases. MEK2 is a dual-specificity kinase that activates the extracellular signal-regulated kinase (ERK) and mitogen-activated protein (MAP) kinase upon agonist binding to receptors. MEK2 plays a key role in the Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathways (1). Approximately 30% of all human cancers have a constitutively activated MAPK pathway, and constitutive activation of MEK2 results in cellular transformation. The ERK/MAP kinase cascade regulates cell growth and differentiation (2).
Shuichan, X. et al. (1997) Mol. Endocrinol. 11:1618.
Louis-François, B. et al. (2003) Mol. Cell. Biol. 23:4778.
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