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Assay Method Information

Assay Name:  Chip Based Microfluidic Mobility Shift Assay
Description:  All assays were performed in 384 well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, as well as four 8-point serial dilutions of staurosporine as reference compound, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on a Thermo CatX workstation equipped with a Innovadyne Nanodrop Express. Between pipetting steps, tips were cleaned in wash cycles using wash buffer. The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 ul per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 1 mM MgCl2, 3 mM MnCl2, 4 uM ATP, 4 uM peptide (5-Fluo-Ahx-GAPDYENLQELNKK-Amid) (purchased from Biosyntan, Berlin, Germany) and 4.5 ul per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 1 mM MgCl2, 3 mM MnCl2, 4 nM Syk (Syk(2-635) (UniProtKB/Swiss-Prot: KSYK_HUMAN, P43405), produced in-house from insect cells). Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 ul per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Phosphorylated and unphosphorylated peptides were separated using the Caliper microfluidic mobility shift technology.
Affinity data for this assay
 

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Last update November 1, 2007
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