| Solution Information | help | |
| Enzyme: | SUMO-conjugating enzyme UBC9 | |
| inhibitor: | BDBM47952 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay materials: 1) E1, E2, GST-SUMO and His6-RanGap-1 proteins were obtained from the assay provider's laboratory. 2) Assay Buffer: 50mM Tris-HCl pH 7.4, 0.3mM DTT, 10 mM MgCl2, 0.005% Tween-20 3) Nickel Chelate acceptor beads (Cat # 6760140) and Glutathione donor beads (Cat # 6765300) were purchased from PerkinElmer 4) Corning 1536-well, white plates (Cat #3725) uHTS Procedure 1) Dispense 2ul of assay buffer into columns 1 and 2, and 2ul of Mixture 1, containing 12.5 nM E1 and 100 nM His-RanGap-1 in the assay buffer, into columns 3-48 of the 1536-well assay plate. 2) Using a HighRes biosolutions pintool dispense 70nl of 2mM compounds in DMSO to columns 5-48 3) Dispense 70nl of DMSO to columns 1-4. 4) Using Thermo Scientific MultiDrop Combi, dispense 2 uL of Mixture 2, containing 20 mM ATP, 12.5 nM E2 and 30 nM GST-SUMO in assay buffer, to all wells. 5) Incubate for 90 min. at room temperature. 6) Using Thermo Scientific MultiDrop Combi, dispense 1ul of pre-mixed (pre-mixed Ni | |
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