PRODUCTION OF POLYCLONAL ANTISERA THAT RECOGNIZE AND DISTINGUISH BETWEEN THE EXTRACELLULAR DOMAINS OF NEURONAL NICOTINIC ACETYLCHOLINE-RECEPTOR SUBUNITS
S. Neff et al., PRODUCTION OF POLYCLONAL ANTISERA THAT RECOGNIZE AND DISTINGUISH BETWEEN THE EXTRACELLULAR DOMAINS OF NEURONAL NICOTINIC ACETYLCHOLINE-RECEPTOR SUBUNITS, Journal of neurochemistry, 64(1), 1995, pp. 332-339
Ligand-gated ion channels are oligomeric transmembrane proteins that u
sually contain more than one kind of monomer. The variety of monomers
available to participate in oligomer formation and the apparent latitu
de in acceptable monomer combinations allows considerable diversity. M
echanisms for identifying the monomers comprising specific receptors a
re needed. We have generated affinity-purified polyclonal antisera tha
t recognize the extracellular domain of nine neuronal nicotinic acetyl
choline receptor (nAChR) subunits and distinguish between them. We pre
pared these antisera by immunizing rabbits with bacterially expressed
recombinant protein representing the N-terminal extracellular domain o
f each neuronal nAChR subunit followed by affinity purification of ant
ibodies against synthetic peptides corresponding to residues 68-81 of
the alpha 1 subunit. We demonstrate subunit specificity of each affini
ty-purified antisera by western blots of the bacterially expressed pro
tein 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.