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Proceedings Paper

Analysis of molecular assemblies by flow cytometry: determinants of Gi1 and by binding
Author(s): Noune A. Sarvazyan; Richard R. Neubig
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Paper Abstract

We report here a novel application of flow cytometry for the quantitative analysis of the high affinity interaction between membrane proteins both in detergent solutions and when reconstituted into lipid vesicles. The approach is further advanced to permit the analysis of binding to expressed protein complexes in native cell membranes. The G protein heterotrimer signal transduction function links the extracellularly activated transmembrane receptors and intracellular effectors. Upon activation, (alpha) and (beta) (gamma) subunits of G protein undergo a dissociation/association cycle on the cell membrane interface. The binding parameters of solubilized G protein (alpha) and (beta) (gamma) subunits have been defined but little is known quantitatively about their interactions in the membrane. Using a novel flow cytometry approach, the binding of low nanomolar concentrations of fluorescein-labeled G(alpha) i1 (F- (alpha) ) to (beta) (gamma) both in detergent solution and in a lipid environment was quantitatively compared. Unlabeled (beta) $gama reconstituted in biotinylated phospholipid vesicles bound F-(alpha) tightly (Kd 6 - 12 nM) while the affinity for biotinylated-(beta) (gamma) in Lubrol was even higher (Kd of 2.9 nM). The application of this approach to proteins expressed in native cell membranes will advance our understanding of G protein function in context of receptor and effector interaction. More generally, this approach can be applied to study the interaction of any fluorescently labeled protein with a membrane protein which can be expressed in Sf9 plasma membranes.

Paper Details

Date Published: 1 May 1998
PDF: 10 pages
Proc. SPIE 3256, Advances in Optical Biophysics, (1 May 1998); doi: 10.1117/12.307055
Show Author Affiliations
Noune A. Sarvazyan, Univ. of Michigan (United States)
Richard R. Neubig, Univ. of Michigan (United States)

Published in SPIE Proceedings Vol. 3256:
Advances in Optical Biophysics
Joseph R. Lakowicz; J. B. Alexander Ross, Editor(s)

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