| Assay Method Information | |
| | Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay |
| Description: | Enzymatic activity of the class I PI3K isoforms in the presence of the compounds of Table 1 above was measured using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay.The TR-FRET assay can monitor formation of the product 3,4,5-inositol triphosphate molecule (PIP3) as it competed with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidylinositide 3-phosphate product results in a decrease in TR-FRET signal as the labeled fluorophore is displaced from the GRP-1 protein binding site.The PI3K isoforms were assayed under initial rate conditions in the presence of 10 μM ATP, and compounds were tested in 10-dose IC50 mode starting at a concentration of 0.5 μM. Control compound, PI-103, was tested in 10-dose IC50 with 3-fold serial dilution starting at 10 μm.Data are normalized based on negative (DMSO) control. The alpha, beta, delta, and gamma IC50 values were calculated from the fit of the dose-response curves to a four parameter equation. IC50 are reported in units of nM. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |