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| Solution Information | help | |
| Enzyme: | Solute carrier family 12 member 5 | |
| inhibitor: | BDBM43908 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | For KCC2 measurements through [86Rb] uptake assays, KCC2-overexpressing HEK293 cells were briefly trypsinized from 10-cm dishes and plated for 2 hours on 35-mm dishes coated with poly-L-Lysine (0.1 mg/ml, Sigma). Cells were washed once and pre-incubated for 15 min with 1 ml hyposmotic saline solution containing in mM: 120 NaCl, 5 KCl, 2 CaCl2, 0.8 MgSO4, 5 HEPES, 5 glucose, 100 uM ouabain, 500 uM N-ethylmaleimide, pH 7.4 with HCl (260-270 mOsm). After the preincubation period, the medium was aspirated and replaced with 1 ml of similar solution without NEM, but containing 1 uCi/ml [86Rb]. After 15 min uptake, the solution was aspirated and the cells were washed thrice with ice-cold solution. The cells were then lysed for 1 hour with 500 ul 0.25N NaOH and then neutralized with 250 ul glacial acetic acid. A 300 ul aliquot was then added to 5 ml liquid scintillation fluid for counting and a 30 ul aliquot was used for protein assay. A 5 ul aliquot of the uptake solution was also counted as | |
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