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Assay Method Information |
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| | Mobility Shift Assay |
| Description: | Agents: 1-fold kinase buffer without MnCl2: 50 mM HEPES, pH 7.5, 0.0015% Brij-35, 10 mM MgCl2, 2 mM DTT. 1-fold kinase buffer with MnCl2: 50 mM HEPES, pH 7.5, 0.0015% Brij-35, 10 mM MgCl2, 10 mM MnCl2, 2 mM DTT. Termination solution: 100 mM HEPES, pH 7.5, 0.0015% Brij-35, 0.2% Coating Reagent#3, 50 mM EDTA. 2.5-fold kinase solution: The kinase was added to the 1-fold kinase buffer to form a 2.5-fold kinase solution. 2.5-fold substrate solution: FAM fluorescence-labelled polypeptide and ATP were added to the 1-fold kinase buffer to form a 2.5-fold substrate solution. Compounds: The final test concentration of the compound was 10 μM at maximum. Firstly, a 50-fold concentration (i.e. 500 μM) was prepared with 100% DMSO. The compound was diluted with 100% DMSO by 5 folds at 5 concentrations (successively 500 μM, 100 μM, 20 μM, 4 μM, and 0.8 μM). After diluting with 1-fold kinase buffer by 10 folds, 5 μl of the samples were transferred to a 384-well reaction plate. Two negative control wells and two positive control wells were set at each line respectively. Replication was made in a 96-well plate in five concentrations of 5-fold compound. 10% DMSO was added to the positive control well, and 5 μl of EDTA (250 mM) was added to the negative control well. Procedure: To the 384-well reaction plate was added 5-fold compound dissolved in 10% DMSO at 5 μl/well. To the 384-well reaction plate was added 2.5-fold kinase solution at 10 l/well. The plate was incubated at room temperature for 10 mins. To the 384-well reaction plate was added 2.5-fold substrate solution at 10 μl/well. The plate was incubated at 28° C. for a certain period. 25 μl termination solution was added to terminate the reaction. |
| Affinity data for this assay | |
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