| 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 2×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-GSTXL665, 1.66 nM anti-His EuK and Diluted in Stop Buffer] were also prepared. Compounds were dissolved at 200×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 4× 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 4× in Assay Buffer, and 2.5 μL of 4×SAE1 was mixed into each compound-containing well. After incubating for 15 minutes at RT, 5 μL of 2×Reaction Buffer was mixed into every well. Then after another 45 minutes at RT, 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 RT, and again after sitting at RT 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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