Phosphatidylinositol (PI) its phosphorylated derivates, collectively called phosphoinositides, are important second messengers that are critical as signaling molecules 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 5, they have been broadly classified three major families: phosphoinositide 3-kinases (PI3Ks), phosphoinositide 4-kinases (PI4Ks) phosphoinositide phosphate-kinases (PIP5Ks PIP4Ks).

Promega lipid kinase enzymes, substrates 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 ready-to-use lipid kinase substrates. The enzymes are available separately can be purchased as part of the PI3K-Glo™ Class I Profiling Kit, which contains PI3Ks (α, β, γ δ; 5μg each), PIP2:3PS Lipid Kinase Substrate (0.25mg) the ADP-Glo™ Kinase Assay, 1,000 assays. The lipid substrates are supplied as frozen small unilamellar vesicles containing a mixture of phosphatidylinositol (PI) phosphoinositol-4,5-bisphosphate (PIP2) at a 1:3 ratio with phosphatidylserine (PS) as carrier lipid. A substrate composed of PIP2 PS at a 1:3 ratio was optimized to use with class I PI3Ks. A substrate composed of PI PS at a 1:3 ratio was demonstrated to be recognized by the majority of family members 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 PIP2:3PS) with a recombinant enzyme ATP, 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 deplete any remaining ATP, leaving only ADP. Next, a detection reagent is added to simultaneously convert ADP to ATP allow the newly synthesized ATP to be converted to light using a coupled luciferase/luciferin reaction.

特点 - 优点

• Employ Complete Solutions for Class I PI3Ks: 
  Purified human recombinant enzymes with high specific activity.
  Ready-to-use lipid substrate (PI 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 excellent sensitivity result in less ambiguous data.
• Save Time: Homogeneous assay with simple "addread" format.
• Avoid False Hits: The special formulation luminescent signal results in low false-hit rate.
• Save Money: Easily scalable to 1,536-well format, reducing cost per well.