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Assay Method Information |
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| | Kinase Assay |
| Description: | CDK1/CycB (200 ng/measuring point) was incubated for 10 min at 22 C. in the presence of different concentrations of test substances (0 and within the range 0.001-10 uM) in assay buffer [50 mM Tris/HCl pH 8.0; 10 mM MgCl2; 0.1 mM Na ortho-vanadate; 1.0 mM dithiothreitol; 0.5 uM adenosine trisphosphate (ATP); 10 ug/measuring point histone IIIS; 0.2 uCi/measuring point 33P-gamma ATP; 0.05% NP40; 1.25% dimethyl sulphoxide]. The reaction was stopped by adding EDTA solution (250 mM; pH 8.0; 15 ul/measuring point).From each reaction mixture, 15 ul were applied to P30 filter strips (Wallac), and unincorporated 33P-ATP was removed by washing the filter strips three times, for 10 min in each case, in 0.5% phosphoric acid. After drying the filter strips for 1 hour at 70 C, the filter strips were covered with scintillator strips (MeltiLex A, Wallac) and stoved for 1 hour at 90 C. The amount of incorporated 33P (substrate phosphorylation) was determined. |
| Affinity data for this assay | |
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