| Assay Method Information | |
| | The [3H]-methyllycaconitine binding assay |
| Description: | 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 phenylmethylsulphonyl fluoride (PMSF), 0.01% (w/v) NaN3, pH 7.4, 4° C.) at 600 rpm in a glass homogenizer. The homogenate is centrifuged (1000×g, 4° C., 10 min) and the supernatant is removed. The pellet is resuspended (20% w/v) and the suspension is centrifuged (1000×g, 4° C., 10 min). The two supernatants are combined and centrifuged (15 000×g, 4° C., 30 min). The pellet obtained in this way is referred to as the P2 fraction.The P2 pellet is suspended in, binding buffer (50 mM Tris-HCl, 1 mM MgCl2, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, pH 7.4), and the suspension is centrifuged (15 000×g, 4° C., 30 min), twice.The residue is resuspended in binding buffer and incubated in a volume of 250 (amount of membrane protein 0.1-0.5 mg) in the presence of 1-5 nM [3H]-methyllycaconitine 0.1% (w/v). BSA (bovine serum albumin) and various concentrations of the test substance at 21° C. for 2.5 h. Incubation is then carried out in the presence of 1 μM α-bungarotoxin or 100 μM nicotine or 10 μM MLA (methyllycaconitine).The incubation is stopped by adding 4 ml PBS (20 mM Na2HPO4, 5 mM KH2PO4, 150 mM NaCl, pH 7.4, 4° C.) and filtering through type A/E glass fibre filters (Gelman Sciences) which have previously been placed in 0.3% (v/v) polyethyleneimine (PEI) for 3 h. The filters are washed twice with 4 ml of PBS (4° C.), and the bound radioactivity is determined by scintillation measurement. All the assays are carried out in triplicate. The dissociation constant Ki of the test substance was determined from the IC50 of the compounds (concentration of the test substance at which 50% of the ligand bound to the receptor is displaced), the dissociation constant KD and the concentration L of [3H]-methyllycaconitine using the equation Ki=IC50/(1+L/KD). |
| Affinity data for this assay | |
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