NVP-TAE 684 (TAE684) is a excellently effective and specific ALK inhibitor(IC50=3 nM).

ALK:3 nM

In vitro Enzyme Assays.: All in vitro enzyme assays are done at Upstate Biotechnology with the exception of InsR and IGF-1R. To determine the IC50 of TAE684 against InsR and IGF-1R a homogeneous time-resolved fluorescence assay is performed. ATP (10 mM) and 20 mg/ml biotinylated PolyEY (Glu, Tyr 4:1) are combined with 50 nL of serial dilutions of TAE684 (10-500 nM) and 4 ng of InsR enzyme in the presence of the kinase reaction buffer (20 mM Tris譎Cl, pH 7.5/10 mM MgCl2/3 mM MnCl2/1 mM DTT/10 mM NaVO4/0.1 mg/ml of BSA). Assays are incubated for 1 hour at ambient temperature. Reactions are terminated by adding 10 mL of the detection solution containing 50 mM EDTA, 500 mM KF, 0.5 mg/ml of BSA, 5 mg/mL Eu3+ cryptate-labeled anti-phosphotyrosine antibody Mab PT66-K, and 5 mg/mL Streptavidin-XLent. The reaction is incubated for half an hour, and fluorescence signals are read on Analyst GT.

Cells are seeded in 384-well plates (2.5×104 cells per well) and incubated with serial dilutions of TAE684 or DMSO for 2–3 days. Luciferase expression is used as a measure of cell proliferation/survival and is evaluated with the Bright-Glo Luciferase Assay System. IC50 values are generated by using XLFit software.(Only for Reference)