| Solution Information | help | |
| Enzyme: | E3 ubiquitin-protein ligase Mdm2 | |
| inhibitor: | BDBM43475 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Materials DMEM (Cat#11995-081), Fetal Bovine serum (Cat#26140-079), Streptomycin (Cat#15140-155), Trypsin-EDTA (Cat#25200-106) and Zeocin (Cat#R250-01) were all purchased from Invitrogen. SteadyLite HTS gene assay reporter system (Cat#6016989) was purchased from Perkin Elmer. The Luminescence assay was carried out in 384 well, white, tissue-culture treated plates from Greiner (Cat#781080). Breathe-easy membranes (Cat#Z380059) were from Sigma. Assay Cells (wild-type or mutant) were plated and allowed to settle at 37C overnight. The cells were then incubated with different concentrations of compound (final DMSO conc:0.25%) at 37C for 2 hrs. SteadyLite luciferase reagent was added, plates incubated for 30 mins and then read on Envision reader. Dose-response protocol 1.Fill 384 well plate with 24 uL of cells (wild-type or mutant) in DMEM (7500 cells per well) using Wellmate (all columns except 1 and 23) 2.Add 24 uL DMEM to columns 1 and 23 using Wellmate 3. Seal plates with Breathe- | |
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