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| Solution Information | help | |
| Enzyme: | Metabotropic glutamate receptor 4 [Q124R] | |
| inhibitor: | BDBM50293743 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Basic protocol: 1. Cells are plated at a density of 20,000 cells/well 1 day prior to assay in Thallium Assay Media (DMEM + 20mM HEPES, 10% dialyzed FBS, 1mM sodium pyruvate). 2. The following day, media is removed from the cells. 3. 20 ul/well of Fluo Zn dye (0.33 uM) in Assay Buffer (HBSS + 20mM HEPES) is added. 4. The plates are incubated for one hour at RT. 5. Dye is removed from the cells and the cell are washed with Assay Buffer leaving 20uL residual volume. 6. Plates are incubated for 10 minutes at RT. 7. Plates are loaded into a Hamamatsu FDSS. 8. Compounds are tested for selectivity at a 10 uM final concentration (prepared as a 2X stock, 20 ul added per well) and the agonist is added at a 5X concentration (10 ul of agonist added per well, 50 uL total volume). A baseline read is taken for 1.5 seconds, the first addition (compound or DMSO-matched vehicle) occurs at 1.5 seconds and the second addition (agonist) is added at 142 seconds. The read continues for 300 seconds. Data an | |
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