Search and Browse
Download
Enter Data
|
Assay Method Information |
|
| | Inhibition Assay |
| Description: | Rat brain tissue (hippocampus or whole brain) is homogenized in aqueous 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, 4C.) at 600 rpm in a glass homogenizer. The homogenate is centrifuged (1000xg, 4C., 10 min) and the supernatant is removed. The pellet is resuspended (20% w/v) and the suspension is centrifuged (1000xg, 4C., 10 min). The two supernatants are combined and centrifuged (15 000xg, 4C., 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 000xg 4C., 30 min), twice. The residue is resuspended in binding buffer to a concentration of 4 mg/ml and incubated in a volume of 250 ul (amount of membrane protein 0.4 mg) in the presence of 2 nM [3H]-methyllyeaconitine, 0.1% (w/v) BSA (bovine serum albumin). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |
|
Home |
| |
Search |
| |
Deposit |
| |
SiteMap |
| |
About us |
| |
Email us |
| |
Info |
|
©2000 BindingDB. All rights reserved. |
|||||||||||||