| Assay Method Information | |
| | FLT3 Kinase Test |
| Description: | FLT3 is obtained from Invitrogen in 50 mM Tris (pH7.5); 100 mM NaCl; 0.05 mM EDTA, 0.05% NP-40, 2 mM DTT; 50% Glycerol # PV3182; Lot 286671; sequence see below). The enzyme is diluted to 720 nM (35 μg/ml) in enzyme dilution buffer and 10 μl aliquots are stored at −80° C.The activity of FLT3 is measured using the Z′-LYTE assay technology from Invitrogen (#PV3191)MethodThe assay is performed in 384 black plates from Corning (#3676) in a final volume of 10 μl by adding 5 μl of kinase peptide mix and 2.5 μl of compound dilution. The reaction is started by addition of 2.5 μl of the 4×ATP solution.Final concentration in assay: FLT3 2 nM, Tyr2 peptide 4 μM, ATP 470 μM (ATP Km for FLT3)Positive controls are reaction mixtures containing no test compound; negative controls (blanks) are reaction mixtures containing no kinase. As a further control, the phosphopeptide solution is added to wells without kinase (=100% phosphorylation control). The non inhibited kinase reaction will result in a phosphorylation corresponding to 20%-30% of the phosphorylation control.The reaction is performed for 1 h at room temperature before 5 μl of the development solution is added. After a further incubation for 1 h at room temperature 5 μl of the stop reagent is added. The plates are read on a Flex Station II 384 (Molecular Devices).To control for any potential inhibition of the protease present in the development solution, the phosphopeptide is incubated with the development solution in the presence of the highest concentration of the test compound (usually 100 μM or 10 μM). |
| Affinity data for this assay | |
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