Simultaneous Detection of Multiple Proteins that Bind to the Identical Ligand in Supported Lipid Bilayers

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• 作者：Chunming Liu ; Da Huang ; Tinglu Yang ; Paul S. Cremer
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：July 21, 2015
• 年：2015
• 卷：87
• 期：14
• 页码：7163-7170
• 全文大小：514K
• ISSN：1520-6882

文摘

Herein, we developed a new separation-based detection method that is capable of simultaneously identifying multiple competitively binding proteins for the same ligand on supported lipid bilayers (SLBs). This strategy used unlabeled target analyte proteins that bind to fluorescently tagged, lipid-conjugated ligands within the SLB. The protein鈥搇igand binding complexes were then focused under an applied potential to different locations within the SLB based on each protein鈥檚 size and charge. Both protein identity and relative surface concentration information could be obtained, simultaneously. Specifically, the competitive binding of streptavidin and goat anti-biotin for biotin-conjugated lipids was explored. It was found that streptavidin could inhibit the binding of goat anti-biotin antibodies for biotin-cap-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2鈥?,3-benzoxadiazol-4-yl)(biotin-cap-NBD-PE) lipids and that streptavidin more effectively outcompeted the anti-biotin antibody at lower protein concentrations. Also, modulating the chemical composition of the membrane helped control the ultimate focusing position and separation of the streptavidin-bound biotin, anti-biotin-bound biotin, and free biotin-conjugated lipid bands. The assay developed herein provides a simple and convenient strategy for simultaneously monitoring target analytes that bind to the identical ligand and may ultimately be useful in developing assays that help overcome problems associated with cross-reactivity.