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Encompassing Amino Acids: full length
Applications: Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.
Assay Conditions: Kinase activity was measured using the ADP-Glo™ Kinase Assay Kit (Promega; Cat# V9101) which quantifies the amount of ADP produced. The ADP-Glo™ Reagent is added to terminate the reaction and deplete the remaining ATP. The Kinase Detection Reagent is then added to convert ADP to ATP and to measure the newly synthesized ATP using a luciferase reaction.PKN3/PRK3 activity was measured by using RSK substrate (KRRRLSSLRA) diluted in water to a final concentration of 1 mg/ml. Reaction was initiated by mixing increasing amounts of the PKN3/PRK3 with 25 µM ATP in 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 0.1 mg/ml BSA prepared with 250 µM DTT and the 20 µg/ml substrate.After a 40-minute incubation at 37°C, the reaction was terminated by addition of the AMP-Glo™ Reagent followed by a subsequent 40-minute incubation at room temperature. Kinase Detection Reagent was then added and incubated for another 30 minutes. Detection of luminescence was measured using the Luminescence Module Protocol on GloMax®-Multi Microplate reader. The corrected activity (RLU) was calculated by removing the blank value for each sample divided by the (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 working solution with an equal volume of Kinase Dilution Buffer IX (1X).
Description: Recombinant human PKN3 also known as PRK3 (protein kinase N3), full length. This construct contains an N-terminal GST-tag. The recombinant protein was affinity purified and kinase active.
Format: Aqueous buffer solution
Formulation: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM glutatione, 0.1 mM EDTA, 0.25 mM DTT, 0.1 mM PMSF, 25% glycerol
Genbank: NM_013355
Host Cell Line: Sf9 cells
Purity: ≥70%
Storage Stability: At least 6 months at -80°C.
Tags: N-terminal GST-tag
Uniprot: Q6P5Z2
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
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