| Assay Method Information | |
| | Mobility-Shift Assay |
| Description: | In this experiment, the inhibitory effects of small molecule inhibitors on 17 kinases were examined by using fluorescent microfluidic mobility shift assay (Mobility-Shift Assay).1. Buffer configuration: 50 mM HEPES, pH 7.5, 0.00015% Brij-35.2. Compounds were configured in 100% DMSO in a concentration gradient and diluted with buffer to 10% DMSO, and added to 384-well plates. Compounds starting at 500 nM are prepared in 100% DMSO to 25 μM and diluted in a gradient of 10 concentrations, then diluted 10-fold in buffer to make an intermediate dilution of the compound containing 10% DMSO and transferred 5 μl to a 384-well plate.3. The kinase was diluted to optimal concentration with the following buffers: 50 mM HEPES, pH 7.5, 0.00015% Brij-35, 2 mM DTT (final concentration of enzyme reaction: VEGFR-1 (FLT1): 2 nM; VEGFR-2 (KDR): 1.2 nM; VEGFR-3 (FLT4): 1.5 nM; FGFR1: 2 nM); FGFR2: 9 nM; FGFR3: 8 nM; FGFR4: 10 nM; PDGFRα: 3.5 nM; c-MET: 10 nM; RET: 7 nM; EGFR: 6 nM). Transfer 10 μl into a 384-well plate and incubate with the compounds for 10 min.4. The substrate was diluted to the optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 0.00015% Brij-35. where the final concentration of the reaction was as follows:VEGFR1 (FLT1): 3 μM Peptide30 (5-FAM-KKKKEEIYFFF-CONH2), 278 μM ATP, 10 mM MgCl2;VEGFR2 (KDR): 3 μM Peptide22(5-FAM-EEPLYWSFPAKKK-CONH2), 92 μM ATP, 10 mM MgCl2;VEGFR3 (FLT4): 3 μM Peptide30 (5-FAM-KKKKEEIYFFF-CONH2), 84 μM ATP, 10 mM MgCl2;FGFR2: 3 μM Peptide22(5-FAM-EEPLYWSFPAKKK-CONH2), 1.9 μM ATP, 10 mM MgCl2;RET: 3 μM Peptide22(5-FAM-EEPLYWSFPAKKK-CONH2), 23 μM ATP, 10 mM MgCl2 5. Read the conversion rate with Caliper Reader and calculate the conversion rate as suppression, formula Percent inhIcition=(max−conversion)/(max−min)*100.6. Calculate the IC50 formula Y=Bottom+(Top−Bottom)/(1+(IC50/X){circumflex over ( )}HillSlope) by fitting it with XL-fit 5.4.0.8 software. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |