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Assay Method Information |
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| | FLT3 Kinase Biochemical Assay |
| Description: | i. Enzyme The assay was performed using FLT3 cytoplasmic domain product and purified in house as GST fused protein. The FLT3 protein (1 microM) was pre activated with 800 microM ATP for 1 hour at 28° C. in order to obtain a linear kinetic.ii. FLT3 Kinase Buffer (KB) Kinase buffer was composed of 50 mM HEPES pH 7.9 containing 4 mM MgCl2, 1 mM DTT, 10 microM Na3VO4, and 0.2 mg/mL BSAiii. Assay Conditions The FLT3 kinase assay was run with a final pre activated enzyme concentration of 2 nM, in the presence of 254 microM ATP (residual ATP from KIT pre activation step is negligible), 8 nM 33P-γ-ATP and 55 microM of substrate BioDB n*24 (Aminoacidic sequence: GGKKKVSRSGLYRSPSMPENLNRPR SEQ ID NO: 1). The peptide was purchased from American Peptide Company (Sunnyvale, Calif.). |
| Affinity data for this assay | |
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