Oblas B, Boyd N D, Singer R H
Anal Biochem. 1983 Apr 1;130(1):1-8. doi: 10.1016/0003-2697(83)90641-3.
A nitrocellulose-gel transfer technique has been adapted to study the interaction of a polypeptide ligand with individual receptor subunits. The acetylcholine receptor isolated from Torpedo californica has been separated into its subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred in a renaturing environment to nitrocellulose sheets. The sheets were incubated with 125I-alpha-bungarotoxin and autoradiographed. A single receptor polypeptide, the alpha subunit (40K) bound the labeled toxin. This binding was demonstrated to be both saturable and specific, although the affinity of 125I-alpha-bungarotoxin (KD, 165 nM) and the potency of d-tubocurarine to displace this binding (IC50, 1 mM) were both reduced by several orders of magnitude when compared to the native receptor.
一种硝酸纤维素凝胶转移技术已被用于研究多肽配体与单个受体亚基之间的相互作用。从加州电鳐分离出的乙酰胆碱受体通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳被分离成其亚基，并在复性环境中转移到硝酸纤维素膜上。将膜与125I-α-银环蛇毒素一起孵育并进行放射自显影。单个受体多肽，即α亚基（40K）结合了标记的毒素。尽管与天然受体相比，125I-α-银环蛇毒素的亲和力（KD，165 nM）和d-筒箭毒碱取代这种结合的效力（IC50，1 mM）都降低了几个数量级，但这种结合被证明是可饱和的且具有特异性。
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