Neff S, Dineley-Miller K, Char D, Quik M, Patrick J
Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030.
J Neurochem. 1995 Jan;64(1):332-9. doi: 10.1046/j.1471-4159.1995.64010332.x.
Ligand-gated ion channels are oligomeric transmembrane proteins that usually contain more than one kind of monomer. The variety of monomers available to participate in oligomer formation and the apparent latitude in acceptable monomer combinations allows considerable diversity. Mechanisms for identifying the monomers comprising specific receptors are needed. We have generated affinity-purified polyclonal antisera that recognize the extracellular domain of nine neuronal nicotinic acetylcholine receptor (nAChR) subunits and distinguish between them. We prepared these antisera by immunizing rabbits with bacterially expressed recombinant protein representing the N-terminal extracellular domain of each neuronal nAChR subunit followed by affinity purification of antibodies against synthetic peptides corresponding to residues 68-81 of the alpha 1 subunit. We demonstrate subunit specificity of each affinity-purified antisera by western blots of the bacterially expressed protein and immunoblot against peptide. We further used these antibodies to demonstrate expression of neuronal nAChR subunits on the surface of transiently transfected simian kidney (COS-7) cells.
配体门控离子通道是寡聚跨膜蛋白，通常包含不止一种单体。参与寡聚体形成的单体种类繁多，且在可接受的单体组合方面似乎具有很大的自由度，这使得其具有相当大的多样性。因此需要鉴定构成特定受体的单体的机制。我们已经制备了亲和纯化的多克隆抗血清，该抗血清可识别九种神经元烟碱型乙酰胆碱受体（nAChR）亚基的细胞外结构域，并能区分它们。我们通过用细菌表达的重组蛋白免疫兔子来制备这些抗血清，该重组蛋白代表每个神经元nAChR亚基的N端细胞外结构域，然后针对与α1亚基第68 - 81位残基对应的合成肽进行抗体的亲和纯化。我们通过对细菌表达蛋白的蛋白质免疫印迹和针对肽的免疫印迹来证明每种亲和纯化抗血清的亚基特异性。我们进一步使用这些抗体来证明神经元nAChR亚基在瞬时转染的猴肾（COS - 7）细胞表面的表达。
