Development of surface-based assays for transmembrane proteins: Selective immobilization of functional CCR5, a G protein-coupled receptor
Section snippets
Materials and methods
Avidin and steptavidin were purchased from Sigma–Aldrich Chemical (St. Louis, MO, USA), biotinylated IgG antibodies were obtained from Chemicon International (Temecula, CA, USA), and the anti-human integrin β1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). n-Undecylenic bromide was purchased from Pfaltz & Bauer (Waterbury, CT, USA). All other chemicals were purchased from Aldrich Chemical (Milwaukee, WI, USA). Tetrahydrofuran and hexamethylphosphoramide were dried
Results and discussion
Fig. 1A shows the SPR shifts obtained for the activation of the initial template by sequential immobilization of avidin, a biotin-conjugated goat anti-mouse IgG (Bt-IgG), and rho 1D4, an anti-rhodopsin antibody that recognizes the modified carboxyl-terminal region on CCR5. Immobilization and saturation are rapid for all steps, with essentially diffusion-limited kinetics for avidin. The kinetics of the Bt-IgG adsorption is slower than that of avidin, presumably due to its larger size (Bt-IgG ∼ 150
Conclusions
Highly reproducible selective attachment of membrane vesicles containing heterologously expressed functional CCR5 and endogenous integrin has been demonstrated using an initial template containing 1–5% biotin in a protein-resistant matrix. Selectivity of membrane vesicle immobilization is accomplished by sequential treatment with avidin or streptavidin, a biotinylated antibody, and a receptor (protein)-specific antibody treatment of the initial template. Considering the complexity of the
Acknowledgments
This work was supported in part by National Institutes of Health grant EY13286 to Kevin Ridge. Portions of this work were presented by Evan Karlik at the 2003 Montgomery County (MD) Area Science Fair (Life Sciences Grand Prize recipient) and at the 2003 International Science and Engineering Fair (Cleveland, OH). Karlik was also a semifinalist in the 2003 Intel Science Talent Search. Certain commercial equipment, instruments, and/or materials are identified in this article to specify the
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Present address: University of Texas Health Science Center-Houston, Center for Membrane Biology, Department of Biochemistry and Molecular Biology, Houston, TX 77030, USA.