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Assay Method Information

Assay Name:  LanthaScreen Kinase Activity Assay
Description:  The enzymatic mTOR activity was measured using a LanthaScreen kinase activity assay (Invitrogen). The enzyme was purchased from Invitrogen (PV4754), as well as the GFP-labelled substrate (4EBP1-GFP; PV4759) and the Tb-antip4EBP1(pThr46) antibody (PV4757). The assay was performed in 50 Mm HEPES buffer, pH 7.5, containing 1.5 mM MnCl2, 10 mM MgCl2, 1 mM EGTA, 2.5 mM DTT and 0.01% Tween-20. The concentration of the assay components were the following: 0.24 nM mTOR kinase, 400 nM 4EBP1-GFP, 10 mM ATP and serial dilutions of the compound (inhibitor) to be evaluated. After 1 h incubation at room temperature, 20 mM EDTA was used to stop the reaction and terbium-labelled antibody (4 nM) added to detect phosphorylated product. The antibody associates with the phosphorylated product resulting in an increased TR-FRET value. The TR-FRET value (a dimensionless number) was calculated as the ratio of the acceptor signal (GFP, emission at 520 nm) to the donor signal (terbium, emission at 495 nm).
Affinity data for this assay
 

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Last update November 1, 2007
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