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| Solution Information | help | |
| Enzyme: | Induced myeloid leukemia cell differentiation protein Mcl-1 | |
| inhibitor: | BDBM33185 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Positives from the primary screen, AID 1022, were selected for follow-up in this confirmatory assay. Reagents: 1. Assay buffer: 20 mM Tris buffer, pH 7.5, 50 mM NaCl, and 0.01% NP40 2. Mcl-1 protein: 6xHis-tagged Mcl-1 protein 3. Terbium-anti-His-Tag antibody from Invitrogen corporation. Procedure: 1. Make assay reaction buffer for uHTS that contains Mcl-1 protein (62.5 nM, final concentration), Noxa-rhodamine peptide (125 nM, final concentration), and terbium-anti-His-Tag antibody (2 nM, final concentration). 2. Dispense 4.5 uL of assay reaction buffer to 1536-well black assay plate. 3. Add library compound (1 mM in DMSO) to each well and incubate plates at room temperature for 2 hr. 4. Record FRET signals with an Envision Multilabel plate reader (Perkin Elmer Life Sciences) with laser. An laser excitation at 340 nm and emission filters at 545 nm and 572 nm are used with a dural dichroic mirror of D400. Data analysis: 1. FRET signals are expressed as FRET ratios: FRET = F572 | |
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