| Solution Information | help | |
| Enzyme: | Kappa-type opioid receptor | |
| inhibitor: | BDBM42532 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay materials: 1) OPRK1 beta-Arrestin (DiscoveRx) 2) Assay Medium: Opti-MEM Medium supplemented with 1% hiFBS, 1X Pen/Strep/Glu, 125 ug/mL Hygromycin (1/2 recommended), 250 ug/mL Geneticin (1/2 recommended) 3) Growth Medium: MEM supplemented with 10% hiFBS, 1X Pen/Strep/Glu, 125 ug/mL Hygromycin (1/2 recommended), 250 ug/mL Geneticin (1/2 recommended) uHTS protocol: Day 1 1) Harvest cells using Enzyme-Free Dissociation Buffer (Invitrogen Cat#13151-14). Add 500 cells/well in 5 uL of media to each well of a white, 1536 well plate. 2) Spin cells at 500 rpm for 1 min, then wrap plates in Saran Wrap. 3) Incubate overnight at 37C with 5% CO2. Day 2 1) Using a Highres Biosolutions pintool pin 30 nL to wells. Columns 1-4 should be DMSO only (Control wells), Columns 5-48 contain test compounds (10uM final in well concentration). 2) Immediately following pintool addition, add 1.0 uL of assay media to columns 1-2 and 1.0 uL of assay media containing 240 nM dynorphin A for a final assay concen | |
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