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H. A. Gussin, F. T. Khasawneh, A. Xie, H. Qian, G. C. Le Breton, D. R. Pepperberg; Characterization of a Novel Polyclonal Anti Human 1 GABAC Antibody. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1288.
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© ARVO (1962-2015); The Authors (2016-present)
GABAC receptors mediate inhibitory synaptic signaling at multiple locations within the retina. Reagents with high affinity for the GABAC extracellular domain will be important for developing the anchoring component of GABAC-modulating chemical structures (e.g., Gussin et al. (2006) J. Amer. Chem. Soc. 128:15701-15713). We have characterized a new polyclonal antibody (here termed GABAC Ab N-14) directed against a peptide of the ρ1 GABAC extracellular domain.
A 14-mer peptide (N-14) within the N-terminal region of the human ρ1 subunit was chosen as the target for antibody production in guinea pig. Peptide synthesis, immunization of animals, and serum collection were contracted to an outside source. The IgG fraction was isolated by Protein-A purification. The antibody was further purified by affinity chromatography using the N-14 cognate peptide.
Affinity-purified GABAC Ab N-14 was used at a 1:10,000 dilution for Western blots, to probe preparations from ρ1 GABAC-expressing neuroblastoma cells and Xenopus oocytes, and from non-expressing (control) cells. Bands at MW ~55 kDa (consistent with the ρ1 subunit MW) were present in lanes from expressing cells, but not in lanes from non-expressing cells. No bands were observed when GABAC Ab N-14 was either excluded or pre-absorbed with N-14 peptide. N-14 spotted on the membrane showed reactivity when probed with GABAC Ab N-14. Analysis of rat retina, baboon retina and rat brain showed a band at ~55 kDa when probed with the antibody. Flow cytometry revealed a rightward shift in the mean fluorescence intensity of GABAC-expressing neuroblastoma cells probed with affinity-purified GABAC Ab N-14, as compared with either (i) non-expressing cells probed with this antibody, or (ii) GABAC-expressing cells probed with control IgG. Specifically, when the GABAC-expressing cells were probed with affinity-purified GABAC Ab N-14 at 1:50 to 1:1,000 dilution, the shift corresponded with the presence of approximately 63 to 47% positive cells. Immunofluorescence labeling (1:2,000 dilution of Protein-A purified GABAC Ab N-14) of live neuroblastoma and HEK cells yielded fluorescence only for GABAC-expressing cells. GABAC-expressing cells incubated without this IgG did not show measurable fluorescence.
GABAC Ab N-14 exhibits high affinity for ρ1 GABAC receptors. The results encourage study of the GABAC subunit specificity of this antibody and further study of its activity in retinal tissue.
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