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| | Kinetics Assay for IC50 Determination |
| Description: | IC50 determinations for cSrc kinases were measured with the HTRF KinEASE-TK assay from Cisbio according to the manufacturer instructions. A biotinylated poly-Glu-Tyr substrate peptide was phosphorylated by cSrc. After completion of the reaction, an anti-phosphotyrosine antibody labeled with europium cryptate and streptavidin labeled with the fluorophore XL665 were added. The FRET between europium cryptate and XL665 was measured to quantify the phosphorylation of the substrate peptide. Kinase, substrate peptide, and inhibitor were preincubated for 2 h before the reaction was started by addition of ATP. A Tecan Safire2 plate reader was used to measure the fluorescence of the samples at 620 nm (Eu-labeled antibody) and 665 nm (XL665 labeled streptavidin) 60 us after excitation at 317 nm. The quotient of both intensities for reactions made with eight different inhibitor concentrations was fit to a Hill four-parameter equation to determine IC50 values. Each reaction was performed in duplicate, and at least three independent determinations of each IC50 were made. |
| Affinity data for this assay | |
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