期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 135, 期 8, 页码 3104-3111出版社
AMER CHEMICAL SOC
DOI: 10.1021/ja3102133
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资金
- DAAD [D/08/46093]
- Council for Chemical Sciences of The Netherlands Organization for Scientific Research (NWO-CW) [700.58.443]
- European Research Council [259183 Sumoman]
- Dutch Ministry of Economic Affairs, Agriculture and Innovation
We report the fabrication of a patterned protein array using three orthogonal methods of immobilization that are detected exploiting a fluorogenic surface. Upon reaction of thiols, the fluorogenic tether reports the bond formation by an instantaneous rise in (blue) fluorescence intensity providing a means to visualize the immobilization even of nonfluorescent biomolecules. First, the covalent, oriented immobilization of a visible fluorescent protein (TFP) modified to display a single cysteine residue was detected. Colocalization of the fluorescence of the immobilized TFP and the fluorogenic group provided a direct tool to distinguish covalent bond formation from physisorption of proteins. Subsequent orthogonal immobilization of thiol-functionalized biomolecules could be conveniently detected by fluorescence microscopy using the fluorogenic surface. A thiol-modified nitrilotriacetate ligand was immobilized for binding of hexahistidine-tagged red-fluorescing TagRFP, while an appropriately modified biotin was immobilized for binding of Cy5-labeled streptavidin.
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