| Assay Method Information | |
| | UBA6 AlphaScreen Assay |
| Description: | The UBA6 enzmatic reaction totals 20 μL and contains 50 mM HEPES (pH 7.5), 0.05% BSA, 0.02% Tween 20, 0.1 mM TCEP, 5 mM MgCl2, 2 μM ATP, 60 nM Flag-Ubiquitin, 20 nM Biotin-his-Use1, and 0.15 nM recombinant human his-UBA6 enzyme. The enzymatic reaction mixture, with and without compound inhibitor, is incubated at 24° C. for 90 minutes in a 384 well plate before termination with 10 μL of Stop/Detection buffer (50 mM HEPES (pH 7.5), 0.05% BSA, 0.02% Tween 20, 0.1 mM TCEP, 20 mM EDTA, 30 μg/ml anti-FLAG Acceptor beads (Perkin Elmer), and 6 μg/mL streptavidin donor beads (Perkin Elmer). After incubation for 2 hours at 24° C. in a dark room, quantification of the AlphaScreen signal is performed on the Pherastar (BMG). |
| Affinity data for this assay | |
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