Production of Polyclonal Antisera that Recognize and Distinguish Between the Extracellular Domains of Neuronal Nicotinic Acetylcholine Receptor Subunits

Abstract

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 olígomer 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 a1 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.

Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures

“…(2001)]. Similarly, immunoblotting experiments with a number of affinity‐purified polyclonal antibodies showed that they reacted only with their homologous bacterially expressed proteins and synthetic peptides [e.g., Neff et al. (1995); Chen and Patrick J (1997)].…”

“…Western blot analyses of nAChR subunits expressed in oocytes and transfected fibroblasts (M10) demonstrated that numerous monoclonal antibodies (e.g., a4: mAb299; a7: mAb319; b2: mAb270, mAb290) do not cross-react with other nAChR subunits [e.g., Conroy et al (1992); Wang et al (1996);Schröder et al (2001)]. Similarly, immunoblotting experiments with a number of affinitypurified polyclonal antibodies showed that they reacted only with their homologous bacterially expressed proteins and synthetic peptides [e.g., Neff et al (1995); Chen and Patrick J (1997)]. Moreover, cell lines such as human neuroblastoma IMR32 (Gotti et al 1995) and SH-SY5Y (Martin-Ruiz et al 1999;Guan et al 2000), human embryonic kidney HEK293 cells (Sallette et al 2005) as well as mouse fibroblast cell line M10 (Guan et al 2000) were immunolabeled with nAChR antibodies -among which were Ab-607, sc-1771 (both a3), mAb299, sc-1772, NRa4-1.5 (all a4); mAb306, sc-1447 (both a7) and sc-1449 (b2)-when stably expressing the respective nAChR subtype, but not when untransfected.…”

Amino Acid Determinants of α7 Nicotinic Acetylcholine Receptor Surface Expression

“…Samples were resolved with SDS-polyacrylamide gel electrophoresis (4% stacking, 8% separating gels) transferred to Immobilon membrane (Millipore, Bedford, MA), and immunodetected with antibodies specific for the extracellular domain of the ␣7 nAChR subunit (31,32). All antibodies were diluted in PBS, 0.1% Tween 20, and 5% nonfat dry milk.…”

The cellular model for Alzheimer's disease research: PC12 cells

“…The capsid is comprised of the major capsid protein VP1 and the minor capsid proteins VP2 and VP3 (Watanabe et al, 2013 )]. The α3 nAChRs, α4 nAChRs and β2 nAChRs have been shown to be expressed on the surface of COS-7 cells (Neff et al, 1995 ). In addition to these common cells, other cells are also adopted in AD research, such as NT2-N, which is derived from the human teratocarcinoma cell line NT2 (ATCC ® CRL-1973 TM ).…”