Affinity purification of nicotinic acetylcholine receptor from rat brain

Identifying the Lipid−Protein Interface of the α4β2 Neuronal Nicotinic Acetylcholine Receptor: Hydrophobic Photolabeling Studies with 3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine

“…This avoids copurification of other neuronal nAChRs, maintains a fixed subunit stoichiometry, and preserves receptor posttranslational modifications. (ii) Although earlier studies reported high levels of receptor enrichment (e.g., 13000-fold enrichment (34) and 7000-13000-fold enrichment (33)), the specific activity of the purified receptor was at best 0.4 nmol of [ 3 H]ACh binding site/mg of protein, and the reported protein yields were either at the microgram level (20 µg; 34) or below the level of detection (33). Furthermore, the yields from rat brain were low and variable (4-20%), suggesting that different populations of neuronal nAChR subtypes were purified depending on the type of column used (33).…”

“…Human α4β2 nAChRs were affinity-purified using a bromoacetylcholine bromide-derivatized Affi-Gel 10 column (see the Experimental Procedures). This ACh-affinity column has been used to purify Torpedo nAChRs ( , ), but its application to the purification of neuronal nAChRs has been very limited ( , ). A number of factors, including the low abundance of neuronal nAChRs in native tissues (<1 pmol/mg) as well as the heterogeneity of nAChR subtypes, have all contributed to the scarcity of reports involving neuronal nAChR purification.…”

Nicotine‐induced phosphorylation of extracellular signal‐regulated protein kinase and CREB in PC12h cells

“…Male Wistar rats (200∼250 g) were used for the [ 3 H]αBgt binding experiments. Preparation of the membrane fraction was essentially as described previously (Nakayama et al . 1990) except for the above homogenizing buffer (buffer A) and suspending buffer (buffer B).…”