| Assay Method Information | |
| | Binding Assay |
| Description: | The [3H]-methyllycaconitine binding assay is a modification of the method described by Davies et al. in Neuropharmacol. 1999, 38, 679-690.Rat brain tissue (hippocampus or whole brain) is homogenized in homogenization buffer (10% w/v, 0.32 M sucrose, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 0.01% (w/v) NaN3, pH 7.4, 4 C.) at 600 rpm in a glass homogenizer. The homogenate is centrifuged (1000xg, 4 C., 10 min) and the supernatant is removed. The pellet is resuspended (20% w/v) and the suspension is centrifuged (1000xg, 4 C., 10 min). The two supernatants are combined and centrifuged (15 000xg, 4 C., 30 min). The pellet obtained in this way is referred to as the P2 fraction.The P2 pellet is washed with binding buffer (50 mM Tris-HCl, 1 mM MgCl2, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, pH 7.4), and centrifuged (15 000x g, 4 C., 30 min), twice.The P2 membranes are resuspended in binding buffer and incubated in a volume of 250 ul (amount of membrane protein 0.1-0.5 mg). |
| Affinity data for this assay | |
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