| Assay Method Information | |
| | ULight-LANCE Assay |
| Description: | Full sequence FAK was purchased from Cell Signaling (Catalog No.: 7796). ULight-poly GT (PerkinElmer #TRF0100-D), Eu-anti-phospho-Tyr (PT66) (PerkinElmer #AD0068) and Lance detection buffer (PerkinElmer #CR-97-100) were purchased from PerkinElmer. The kinase solution (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, 0.01% Tween-20) was adjusted to a final concentration of 3 nM by adding FAK diluted to 6 nM (2×) in white 384 OptiPlate, and added with an amount of 5 μL. 4× ULight-poly GT was adjusted to a final concentration of 100 nM. ATP (Sigma #A2383) was adjusted to a final concentration of 10 μM and in 2.5 μL aliquots. The test compound was sequentially diluted at 12 concentrations and treated with an amount of 0.5 μL. After shaking well and allowing to react at room temperature for 60 minutes, 5 μL of ethylenediaminetetraacetic acid (EDTA, final concentration 40 mM) diluted in Lance detection buffer (Lance detection buffer) was added and the mixture was left at room temperature for 5 minutes to stop the reaction. After adding 4× Eu-anti-phospho-Tyr (PT66) phosphorylated antibody diluted to a final concentration of 2 nM in detection buffer with an amount of 5 μL, and reaction was performed at room temperature for 60 minutes. After adjusting to detect time-resolved fluorescence energy transfer (TR-FRET) at excitation wavelength 320 nm and emission wavelength 665 nm, signals were detected using EnVision Multilabel Reader. |
| Affinity data for this assay | |
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