| Solution Information | help | |
| Enzyme: | Short transient receptor potential channel 4 | |
| inhibitor: | BDBM79622 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay overview: The objective of this assay is to validate compounds that inhibit the G-protein receptor coupled activation of the TRPC4 cation channel. This assay employs automated electrophysiology to measure currents in HEK293 cells that express the TRPC4beta and mu-Opioid receptor with voltage clamp protocols in the presence or absence of test compounds. Solutions The Cs-Asp internal solution contained (in mM): 150 Cs-aspartate, 2 MgCl2, 0.36 CaCl2, 1 EGTA, 4 MgATP, 0.3 NaGTP, and 10 HEPES, pH 7.20 with CsOH (calculated free [Ca2+] = 100 nM) The external solution contained (in mM): 150 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 d-glucose, pH 7.40, with NaOH. Assay Protocol 1.Cell culture: Cells were routinely cultured in DMEM/high glucose medium, supplemented with 10% Heat Inactivated Fetal Bovine Serum (HiFBS), 50 IU/ml penicillin, 50 ug/mL streptomycin, 500 ug/mL G418 and 40 ug/mL hygromycin. 2.TRPC4 cells were grown in to 80-90% confluency and dissociated with Detachin | |
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