Phosphatidylinositol (PI) and its phosphorylated derivates, collectively called phosphoinositides, are important second messengers that are critical as signaling molecules and for cellular membrane remodeling. These derivatives are generated by a family of kinases called phosphoinositide lipid kinases (PIKs). Nineteen PIK isoforms have been identified in mammals. Based on their ability to preferentially phosphorylate the hydroxyl group of the inositol ring on position 3, 4 or 5, they have been broadly classified into three major families: phosphoinositide 3-kinases (PI3Ks), phosphoinositide 4-kinases (PI4Ks) and phosphoinositide phosphate-kinases (PIP5Ks and PIP4Ks). Promega lipid kinase enzymes, substrates and detection 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 reagents include purified human recombinant proteins of Class I PI3Ks, optimized reaction buffer and ready-to-use lipid kinase substrates. The enzymes are available separately or can be purchased as part of the PI3K-Glo Class I Profiling Kit, which contains PI3Ks (alpha, beta, gamma and delta; 5ug each), PIP2:3PS Lipid Kinase Substrate (0.25mg) and the ADP-Glo Kinase Assay, 1 000 assays. The 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 was 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 principle of the lipid kinase assay is illustrated in Figure 1. 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. After the kinase reaction, an ATP-depletion reagent is added to terminate the lipid kinase reaction and deplete any remaining ATP, leaving only ADP. Next, 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.|
Employ Complete Solutions for Class I PI3Ks:
Purified human recombinant enzymes with high specific activity.
Ready-to-use lipid substrate (PI or PIP2).
Universal reaction buffer formulation.
Highly sensitive detection assay.
Observe Excellent Selectivity: High signal-to-background ratios even at low % conversion of substrate.
Obtain Reliable Results: The broad dynamic range, low background and excellent sensitivity result in less ambiguous data.
Save Time: Homogeneous assay with simple "add-and-read" format.
Avoid False Hits: The special formulation and luminescent signal results in low false-hit rate.
Save Money: Easily scalable to 1,536-well format, reducing cost per well.