| Assay Method Information | |
| | HTRF Assay |
| Description: | As reagents for the kinase reaction itself and the quantification of the reaction product, the PI3-Kinase HTRF Assay kit from Millipore (#33-017) was used. With this kit the phosphatidylinositol 3,4,5-trisphosphate (PIP3) generated in the kinase reaction is detected by displacement of a biotinylated ligand from an energy transfer complex consisting of a Europium-labeled anti-GST monoclonal antibody, a GST-tagged PH domain, biotinylated PIP3 and Streptavidin-Allophycocyanin (APC). As kinase a complex of N-terminal His6-tagged recombinant full-length human p110alpha and untagged, recombinant, full length, human p85alpha , coexpressed by baculovirus infected Sf21 insect cells and purified using Ni2+/NTA-agarose, was used (Millipore product #14-602).For the assay 50 nL of a 80-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 3 uL of a solution of PI3Kalpha . |
| Affinity data for this assay | |
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