Brain nicotinic receptors isolated by a monospecific antibody against a synthetic alpha-3 subunit receptor peptide compared to a monoclonal anti-idiotypic (to nicotine) antibody.
Nicotinic cholinergic receptor proteins purified from rat brain by immunoaffinity chromatography were characterized using the anti-S3 polyclonal antibody vs. the anti-idiotypic monoclonal antibody 422F11 (generated against an antibody to nicotine). Anti-S3 IgG was purified to homogeneity; anti-S3-Sepharose 4B and 422F11-Sepharose 4B each depleted 3H-nicotine binding sites from brain. Nicotinic receptors isolated from both immunoaffinity columns showed major bands (silver-stained) at 55K and 70K. Using anti-S3 serum as probe, Western blots of nicotinic receptors isolated by the two immunoaffinity gels also showed major bands at 55 and 70K. However, Western blots of fresh brain extracts revealed a major band at 80K and minor bands at 55K and 70K. These results show similar nicotinic cholinergic receptor proteins isolated by the anti-S3 and 422F11 anti-idiotypic antibodies; 80K was dominant only when fresh brain extract was subjected to Western blotting without prior immunoaffinity purification.
Madhok
T C
TC
Endocrine-Neuroscience Laboratory, Minneapolis Medical Research Foundation, MN.
Bjercke
R J
RJ
Langone
J J
JJ
Sharp
B M
BM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D007130:Immunoglobulin Idiotypes / Q000302:isolation & purification; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009538:Nicotine / Q000276:immunology / Q000378:metabolism*; D010455:Peptides / Q000276:immunology; D051381:Rats; D011978:Receptors, Nicotinic / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007130:Immunoglobulin Idiotypes; D046911:Macromolecular Substances; D010455:Peptides; D011978:Receptors, Nicotinic; D009538:Nicotine