Detection of alpha7 nicotinic acetylcholine receptors with the aid of antibodies and toxins.
Neuronal nicotinic acetylcholine receptors (nAChRs) containing alpha7 subunit are well represented in the brain and some non-neuronal tissues, and their malfunctioning is associated with diverse pathologies. Therefore, detection and quantification of alpha7 nAChR are important tasks. The affinity-purified antibodies were prepared against the 1-23 and 179-190 fragments of the human and rat alpha7 nAChR extracellular domain. The specificity and selectivity of these alpha7 (1-23) and alpha7 (179-190) antibodies was tested by ELISA in model systems: the E. coli-expressed alpha7 subunit extracellular domain and the pituitary cell line GH(4)C(1) stably expressing human alpha7 nAChR. On the rat brain slices two antibodies and biotinylated alpha-cobratoxin specifically stained the hippocampus region known to be rich in alpha7 nAChR. Western blot analysis revealed that in the human thalamus membranes and in rat brain membranes, antibodies alpha7 (1-23) stained a single band of 62 kDa, while the alpha7 (179-190) antibodies stained a doublet of 53-54 kDa. The results obtained show that utilization of model systems and a combination of several antibodies with appropriately labeled toxins may provide better ways for detection of alpha7 nAChR.
Tsetlin
V
V
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia. vits@ibch.ru
Shelukhina
I
I
Kryukova
E
E
Burbaeva
G
G
Starodubtseva
L
L
Skok
M
M
Volpina
O
O
Utkin
Yu
Y
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D019921:Biotinylation; D001923:Brain Chemistry; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006624:Hippocampus / Q000737:chemistry; D006801:Humans; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D051381:Rats; D011978:Receptors, Nicotinic / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D015203:Reproducibility of Results; D013788:Thalamus / Q000737:chemistry; D064569:alpha7 Nicotinic Acetylcholine Receptor
D000906:Antibodies; C579167:Chrna7 protein, human; C579165:Chrna7 protein, rat; D003039:Cobra Neurotoxin Proteins; D010446:Peptide Fragments; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; C025154:alpha-cobratoxin