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Production of Polyclonal Antisera that Recognize and Distinguish Between the Extracellular Domains of Neuronal Nicotinic Acetylcholine Receptor Subunits
Author(s) -
Neff Shawn,
DineleyMiller Kelly,
Char David,
Quik Maryka,
Patrick Jim
Publication year - 1995
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1995.64010332.x
Subject(s) - polyclonal antibodies , nicotinic acetylcholine receptor , antiserum , acetylcholine receptor , protein subunit , extracellular , biochemistry , biology , microbiology and biotechnology , receptor , transmembrane domain , affinity chromatography , chemistry , antibody , gene , immunology , enzyme
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 α1 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.