| Assay Method Information | |
| | Biological Assay |
| Description: | The kinase reaction is performed in a final volume of 50 μl per well of a half area COSTAR, 96 well plate. The final concentrations of ATP and phosphatidyl inositol in the assay are 5 μM and 6 μg/mL, respectively. The reaction is started by the addition of PI3 kinase, e.g. PI3 kinase δ. 10 μl test compound in 5% DMSO per well in columns 2-1. Total activity is determined by addition 10 μl of 5% vol/vol DMSO in the first 4 wells of column 1 and the last 4 wells of column 12. The background is determined by addition of 10 μM control compound to the last 4 wells of column 1 and the first 4 wells of column 12. 2 mL ‘Assay mix’ are prepared per plate: 1.912 mL of HEPES assay buffer 8.33 μl of 3 mM stock of ATP giving a final concentration of 5 μM per well 1 μl of [33P]ATP on the activity date giving 0.05 μCi per well 30 μl of 1 mg/mL PI stock giving a final concentration of 6 g/mL per well 5 μl of 1 M stock MgCl2 giving a final concentration of 1 mM per well 20 μl of the assay mix are added per well. 2 mL ‘Enzyme mix’ are prepared per plate (x* μl PI3 kinase p110β in 2 mL of kinase buffer). The ‘Enzyme mix’ is kept on ice during addition to the assay plates. 20 μl ‘Enzyme mix’ are added/well to start the reaction. The plate is then incubated at room temperature for 90 minutes. The reaction is terminated by the addition of 50 μl WGA-SPA bead (wheat germ agglutinin-coated Scintillation Proximity Assay beads) suspension per well. The assay plate is sealed using TopSeal-S (heat seal for polystyrene microplates, PerkinElmer LAS [Deutschland] GmbH, Rodgau, Germany) and incubated at room temperature for at least 60 minutes. The assay plate is then centrifuged at 1500 rpm for 2 minutes using the Jouan bench top centrifuge (Jouan Inc., Nantes, France). The assay plate is counted using a Packard TopCount, each well being counted for 20 seconds. The volume of enzyme is dependent on the enzymatic activity of the batch in use. |
| Affinity data for this assay | |
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