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| Solution Information | help | |
| Enzyme: | E3 ubiquitin-protein ligase XIAP | |
| inhibitor: | BDBM37302 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Bir1/2 assay materials: 1) Bir1/2 protein and rhodamine-SMAC peptide (AVPIAQK-rhodamine) were provided by Prof. John Reed (Sanford-Burnham Medical Research Institute, San Diego, CA) 2) Assay buffer: 31.25 mM HEPES-NaOH, pH 7.5, 1.25 mM TCEP, 0.00625% Tween 20. 3) Bir1/2 working solution contained 2.5 uM Bir1/2 in the assay buffer. 4) Rhodamine-SMAC working solution contained 50 nM Rhodamine-SMAC in the assay buffer. Bir1/2 HTS protocol: 1) 4 uL of 100 uM compounds in 10% DMSO were dispensed in columns 3-24 of Greiner 384-well black small-volume plates (784076). Columns 1 and 2 were added with 4 uL of 10% DMSO. 2) Positive control wells, that contained no Bir1/2, were assigned to column 1 and were added 8 uL of assay buffer using WellMate bulk dispenser (Matrix). 3) 8 uL of Bir1/2 working solution was added to columns 2-24 using WellMate bulk dispenser (Matrix). Negative control wells that contained no compounds were assigned to column 2. 4) Plates were briefly spinned down and in | |
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