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Encompassing Amino Acids: 1400-2098(end)
Applications: Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.
Assay Conditions: PIP5K3 activity was measured using the ADP-Glo™ Kinase Assay kit (Promega; Cat# V9101) which quantifies the amount of ADP produced by phosphorylation. The ADP-Glo™ Reagent is added to terminate the reaction and quench the remaining ATP. The Kinase Detection Reagent is then added to convert ADP to ATP and to measure the newly converted ATP using a luciferase reaction.Assay: kinase activity was measured using a PIP3:PS substrate (250:2000 µM stock solution, sonicated for 1 min before use). The reaction was initiated by mixing Increasing amounts of the protein kinase with 50 µM ATP (final concentration) in 45 mM HEPES, pH 7.5, 40 mM NaCl, 1.6 mM MgCl2, 0.5 mM EGTA, 20 µg/ml BSA, 40 µM fresh DTT, 0.03% Trition-X100 and PIP3:PS substrate (50:400 µM final). The reaction was terminated by addition of an equal volume of the ADP-Glo™ Reagent supplemented with 10mM MgCl2, and the Kinase Detection Reagent was added. Phosphorylation was measured by detection of luminescence. The blank was determined from a "no kinase" sample.
Description: Recombinant human PIP5K3 (1-phosphatidylinositol 3-phosphate 5-kinase, also known as PIKFYVE), encompassing amino acids 1400-2089(end). The construct contains an N-terminal GST-tag. The protein kinase was affinity purified and is kinase-active.
Format: Aqueous buffer solution
Formulation: 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
Genbank: NM_015040
Purity: 0,85
Storage Stability: At least 6 months at -80°C.
Tags: N-terminal GST-tag
Uniprot: Q9Y2I7
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
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