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Assay Method Information |
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| | Kinase Assay |
| Description: | PI3K KinaseGlo assay: 50 mL of compound dilutions were dispensed onto black 384-well low volume Non Binding Styrene (NBS) plates (Costar Cat. No. NBS#3676). L-a-phosphatidylinositol (PI), provided as 10 mg/ml solution in methanol, was transferred into a glass tube and dried under nitrogen beam. It was then resuspended in 3% OctylGlucoside (OG) by vortexing and stored at 4 C. The KinaseGlo Luminescent Kinase Assay (Promega, Madison/WI, USA) is a homogeneous HTS method of measuring kinase activity by quantifying the amount of ATP remaining in solution following a kinase reaction.5 uL of a mix of PI/OG with the PI3K subtype were added (Table 1). Kinase reactions were started by addition of 5 ul of ATP-mix containing in a final volume 10 uL 10 mM TRIS-HCl pH 7.5, 3 mM MgCl2, 50 mM NaCl, 0.05% CHAPS, 1 mM DTT and 1 uM ATP, and occurred at room temperature. Reactions were stopped with 10 ul of KinaseGlo and plates were read 10 mins later in a Synergy2 reader. |
| Affinity data for this assay | |
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