| Assay Method Information | |
| | Kinase Inhibiting Activity |
| Description: | A reaction buffer (100 mM HEPES (pH 7.4), 10 mM MgCl2, 0.003% Brij-35, 0.004% Tween-20, and 1 mM DTT) was mixed with a RET recombinant protein (RET wild type; Invitrogen # PV3819, final concentration: 80 pg/ul, or a RET Gatekeeper mutation (V804L); Invitrogen # PV4397, final concentration: 80 pg/ul) to prepare a RET kinase solution. The test compound was prepared to have a final concentration of 4000 nm with DMSO, and further, test compound samples at 12 different concentrations were prepared with a dilution magnification of √10. 19 uL of the RET kinase solution was added to each of lines A to P of a 384-well plate, and thereafter, the test compound at each concentration was added to lines C to N, and further, 1 uL of dimethyl sulfoxide (hereinafter referred to as DMSO) was added to each of lines A, B, O and P. Thereafter, the obtained mixtures were each preincubated at room temperature for 20 minutes. Furthermore, a substrate solution A containing ATP (final concentration: 1 mM) and a substrate solution B containing no ATP, both in addition to a reaction buffer and FL-Peptide 22 (PerkinElmer, #760366, final concentration: 1.5 uM) were produced. The substrate solution A was added in an amount of 5 uL to lines B to 0, whereas the substrate solution B was added in an amount of 5 uL to lines A and P. The obtained mixtures were each incubated at 28° C. for 45 minutes. A reaction termination solution (100 mM HEPES (pH 7.4), 0.015% Briji-35, 40 mM EDTA, and 0.1% Coating Reagent 3) was added in an amount of 40 ul to the reaction mixture, so as to terminate the reaction. |
| Affinity data for this assay | |
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