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Assay Method Information |
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| | Biochemical Activity Assay |
| Description: | FLT3(ITD) biochemical assays were performed on two different assay system platforms, using Transcreener ADP Assay FP method (BellBrook Labs, Madison, Wis., US) and IMAP FP biochemical activity assays (Molecular Devices, Sunnyvale, Calif., US).In each case the assays were performed in low protein binding, black, round bottom 384-well plates type 3676 (Corning, One Riverfront Plaza, NY, US). Kinase inhibitor compounds were dissolved in 100% DMSO to 5 mM and then we prepared serial dilutions in order to determine IC50 values.In the TranScreener FLT3(ITD) assay we used the following materials in the following final concentrations for the reaction: 8 nM FLT3(ITD) (ProQinase, Freiburg, Germany), 0.05 mg/ml Poly Glu-Tyr peptide (Sigma-Aldrich, Budapest, Hungary) as a substrate, 20 mM HEPES pH 7.5 (Sigma-Aldrich), 1 mM DTT (Sigma-Aldrich), 3 mM MgCl2 (Sigma-Aldrich), 3 mM MnCl2 (Sigma-Aldrich), and 0.01 V % V Tween20 (Sigma-Aldrich) as a detergent. The kinase reaction had started by the addition of 2 μl 5× enzyme, and the reaction had been progressing in the volume of 10 μl for 1 hour at room temperature. The reaction has been stopped by adding 10 μl Transcreener Stop and Detection Solution and had been incubated for additional 1 hour. The solution contained in every case 20 mM HEPES pH 7.5, 40 mM EDTA, 0.02 V/V % Brij35 and 3 nM ADP Alexa633 Tracer. The ADP antibody concentration was 2.08 μg/ml according to the Kmapp value which was 1 μM. Then fluorescence polarization and fluorescence intensity was measured using Analyst GT Multimode Reader (Molecular Devices).In the IMAP FP assay the reaction conditions were the following: 16-45 nM FLT3(ITD) (ProQinase), 400 nM 5TAMRA-GEEPLYWSFPAKKK-NH2 dyed peptide for substrate (Genecust, Dudelenge, Luxembourg), 20 mM HEPES pH 8, 1 mM DTT, 10 mM MgCl2, 2 mM MnCl2, and 0.01V % V Brij35 detergent (Sigma-Aldrich). The ATP concentration was 5.1 μM. The kinase reaction had started by the addition of 2 μl 5× enzyme, and the reaction had been progressing in the volume of 10 μl for 1 hour at room temperature and then was terminated by adding 10 μl IMAP Detection mixture. Fluorescence polarization and fluorescence intensity was measured using Analyst GT Multimode Reader (Molecular Devices) after 1.5 hour of incubation on room temperature. |
| Affinity data for this assay | |
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