| Assay Method Information | |
| | Kinase Activity Inhibition Assay |
| Description: | Recombinant FGFR1 (2.5 nM), FGFR2 (1 nM), FGFR3 (5 nM), or FGFR4 (12 nM) was prepared as a mixture with substrate KKKSPGEYVNIEFG (SEQ ID NO:1) (20 M, FGFR1 substrate); and Poly [E,Y] 4:1 (0.2 mg/ml, FGFR2,3,4 substrate)] in kinase reaction buffer (20 mM HEPES-HCl, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 1 mM EGTA, 0.02% Brij35, 0.1 mM Na3VO4, 0.02 mg/ml BSA, 2 mM DTT, and 1% DMSO). Compound was added to the enzyme/substrate mixture using acoustic technology (Labcyte Echo 550, Sunnyvale, Calif.)(see, Olechno et al., 2006, Improving IC50 results with acoustic droplet ejection. JALA 11, 240-246) and pre-incubated for 0, 15, or 60 minutes at room temperature. After compound pre-incubation, a mixture of ATP (Sigma-Aldrich ) and 33P- -ATP (PerkinElmer) was added to a final concentration of 10 M to initiate kinase reactions. Reactions were incubated for 120 minutes at room temperature and then spotted onto Whatman P81 ion exchange filter paper. |
| Affinity data for this assay | |
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