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Applications: Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.
Assay Conditions: MKK4 activity was measured by using unactive 0.1 mg/ml JNK1 protein, diluted in 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM glutathione, 0.1 mM EDTA, 0.25 mM DTT, 0.1 mM PMSF and 25% glycerol, in the ADP Glo™ kinase assay (Promega #V9101). Reaction was initiated by mixing increasing amounts of the MKK4 with 25 µM ATP in 40 mM Tris-HCl, pH 7.4, 25 mM MgCl2, 0.1 mg/ml BSA prepared with 50 µM DTT and substrate at a final concentration of 20 µg/ml final concentration.After a 40-minute incubation at Room Temperature (RT), the reaction was terminated by addition of ADP-Glo™ Reagent, followed by a subsequent 40-minute incubation at RT. Kinase Detection Reagent was added, and the reaction was incubated for another 30 minutes at ambient temperature. Detection of luminescence was measured using the Luminescence Module Protocol on GloMax®-Multi Microplate Multimode Reader. The corrected activity (RLU) was calculated by removing the blank value for each sample. The Kinase Specific Activity was calculated as follows: RLU / [(specific activity of ADP in RLU/pmol)*(Reaction time in min)*(Enzyme amount in µg or mg)]. The blank was determined from a "no kinase" sample by replacing the enzyme solution with an equal volume of Kinase Dilution Buffer IX (1X).
Description: Recombinant human full-length MKK4 (dual-specificity mitogen-activated protein kinase kinase 4, also known as MAP2K4 or MAPKK4). This construct contains an N-terminal His-tag. The recombinant protein was affinity purified and is active.
Format: Aqueous buffer solution
Formulation: 50 mM Sodium Phosphate, pH 7.0, 300 mM NaCl, 150 mM Imidazole, 0.25 mM DTT, 25% glycerol
Genbank: NM_003010
Host Cell Line: Sf9 cells
Purity: ≥70%
Storage Stability: At least 6 months at -80°C.
Tags: N-terminal His-tag
Uniprot: P45985
Warnings: Avoid freeze/thaw cycles
Biosafety Level: Not applicable (BSL-1)
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