| Assay Method Information | |
| | SAE High Throughput Biochemical Assay Protocol |
| Description: | Assay buffer was prepared [50 mM HEPES, pH 7.5, 0.1% BSA and 10 mM MgCl2] as was the Stop Buffer [100 mM HEPES, pH 7.5, 0.05% Tween20, and 410 mM KF]. The 2x Reaction Buffer [80 nM SUMO-GST, 80 nM UBC9-His, 200 µM ATP and diluted in Assay Buffer] and Antibody Reaction Mix [13.34 nM anti-GST XL665, 1.66 nM anti-His EuK and Diluted in Stop Buffer] were also prepared. Compounds were dissolved at 200x in DMSO in a 384-well plate. Serial dilutions were performed in DMSO for each compound, and then each concentration was diluted another 50-fold in Assay Buffer, to 4x the final concentration. 2.5 µL of each concentration was transferred into its own well in a 384-well plate. SAE1 was then diluted to 4x in Assay Buffer, and 2.5 µL of 4x SAE1 was mixed into each compound-containing well. After incubating for 15 minutes at room temperature, 5 µL of 2x Reaction Buffer was mixed into every well. Then after another 45 minutes at room temperature, 10 µL of Antibody Reaction Mix was added and mixed into every well. This plate was then read on an HTRF-compatible plate reader after 2 hours at room temperature, and again after sitting at room temperature overnight. [Final Component Concentrations: SAE1: 12.5 nM ; SUMO-GST: 40 nM ; UBC9-His: 40 nM ; Anti-GST-XL665: 6.67 nM ; Anti-His-EuK: 0.83 nM ; and ATP: 100 µM]. |
| Affinity data for this assay | |
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