ADP-Glo™ Lipid Kinase Assay Technical Manual

Literature # TM365

ADP-Glo™ Lipid Kinase Systems provide a complete set of reagents for performing phosphoinositide lipid kinase (PIK) reactions using a luminescent ADP-detection platform, the ADP-Glo™ Kinase Assay. The systems include purified recombinant proteins of class I PI3Ks, optimized reaction buffer and ready-to-use lipid kinase substrates. Lipid substrates are supplied as frozen small unilamellar vesicles containing a mixture of phosphatidylinositol (PI) or phosphoinositol-4,5-bisphosphate (PIP2) at a 1:3 ratio with phosphatidylserine (PS) as carrier lipid. A substrate composed of PIP2 and PS at a 1:3 ratio has been optimized to use with class I PI3Ks. A substrate composed of PI and PS at a 1:3 ratio was demonstrated to be recognized by the majority of family members and provides a universal PI lipid kinase substrate.

The lipid kinase reaction is performed by incubating lipid substrate (PI:3PS or PIP2:3PS) with a recombinant enzyme and ATP, and the kinase activity is measured using the ADP-Glo™ Kinase Assay. The ADP-Glo™ Kinase Assay is performed in two steps. First, after the kinase reaction, an ATP-depletion reagent is added to terminate the lipid kinase reaction and deplete any remaining ATP, leaving only ADP. Second, a detection reagent is added to simultaneously convert ADP to ATP and allow the newly synthesized ATP to be converted to light using a coupled luciferase/luciferin reaction.