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Assay Method Information |
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| | Aurora B Enzyme Assay |
| Description: | Aurora B (2 μM) was preactivated by equivalent concentration of GST-INCENP in 30 mM Tris-HCl pH 8.0, 0.4 mM ATP, 2 mM MgCl2, 0.1 mM EGTA, 0.1% BME (beta mercaptoethanol), 0.1 mM sodium vanadate, 10 mM DTT for 3 hours at 30° C. This solution was then dialysed for 5 hours against 50 mM Tris-HCl, pH 7.5, 270 mM sucrose, 150 mM NaCl, 0.1 mM EDTA, 0.1% BME, 1 mM benzamidine and 0.2 mM PMSF at 4° C. Aurora B/INCENP complex was aliquoted and frozen at −80° C. §Human INCENP (826-919) clone DU930 was received from University of Dundee, it is a GST N-terminal tagged protein. A final concentration of 2 nM of Aurora B/INCENP complex was added to the assay buffer (25 mM HEPES, 25 mM NaCl 0.0025% Tween-20, pH 7.2 0.015% BSA, 1 μM DTT). 3 μl of this solution was added to wells containing 0.1 μl of various concentrations of compound or DMSO vehicle in Greiner low volume 384 well black plate at room temperature for 30 mins. The reaction was initiated by the presence of 3 μl of substrate reagent containing 100 nM 5FAM-PKA-tide (GRTGRRNSI-NH2), 2 M ATP and 2 mM MgCl2 in assay buffer (25 mM HEPES, 25mM NaCl 0.0025% Tween-20, pH 7.2 0.015% BSA, 1 μM DTT) with a final DMSO level of 1.7%. The reaction was incubated for a further 120 mins at room temperature, and then terminated by the addition of 6 μl of a 1:500 dilution Progressive Binding Reagent solution (Part: R7287) in the manufacturers buffer A (Part: R7285) and manufacturers buffer B (Part R7286) and left to incubate for 120 mins at room temperature. The degree of phosphorylation of the 5FAM-PKA-tide (GRTGRRNSI-NH2) was measured using an Acquest plate reader (Molecular Devices, Sunnyvale, US) with excitation 485 nM, emission at 530 nM and using a 505 nmM dichroic lens. |
| Affinity data for this assay | |
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