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Monospecific antibodies as probes for the stoichiometry of recombinant GABA(A) receptors.

Knight, AR; Stephenson, FA; Tallman, JF; Ramabahdran, TV; (2000) Monospecific antibodies as probes for the stoichiometry of recombinant GABA(A) receptors. Receptors Channels , 7 (3) pp. 213-226.

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Abstract

GABA(A) receptors composed of alpha1beta3 gamma2 and alpha1beta3 subunits were expressed in insect Sf9 cells and solubilized in 1% Triton X100. In sucrose density gradients, [3H]-Ro15-1788 binding activity, in the case of alpha1beta3 gamma2, and [3H]-muscimol binding activity, in the case of alpha1beta3 containing receptors sedimented as a single sharp peak suggesting the formation of receptors containing a defined number of subunits. When alpha1beta3gamma2 -containing receptors were incubated with an alpha-subunit specific antibody (bd24), a single class of antibody receptor complex was formed irrespective of the receptor-antibody ratio. This is consistent with two alpha subunits cross-linked within the receptor by the antibody. Similar results were obtained using a beta-subunit specific antibody (bd17). Several classes of antibody-receptor complex were formed when receptors were pre-incubated with a gamma specific antibody (anti gamma(2) 1-15 Cys). This profile is consistent with the presence of a single gamma subunit in each complex. Experiments with alpha1beta3 subunit containing receptors and antibody bd24 produced a profile similar to that seen with alpha1beta3 gamma2 receptors, consistent with two alpha subunits per receptor complex. In this case, the anti-beta subunit antibody, bd17, produced a unique and complex profile consistent with three beta subunits per receptor. This method permits the rapid determination of subunit stoichiometries of homogeneous receptor populations

Type: Article
Title: Monospecific antibodies as probes for the stoichiometry of recombinant GABA(A) receptors.
Location: England
Keywords: Antibodies, Monoclonal, Antibody Specificity, Antigen-Antibody Complex, Epitopes, Flumazenil, GABA Agonists, Humans, Molecular Probes, Muscimol, Protein Conformation, Protein Subunits, Receptors, GABA-A, Recombinant Proteins
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy > SoP Pharmaceutical and Bio Chemistry
URI: http://discovery.ucl.ac.uk/id/eprint/1351149
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