Oriented Protein Immobilization using Covalent and Noncovalent Chemistry on a Thiol-Reactive Self-Reporting Surface

Author

Dorothee Wasserberg, Carlo Nicosia , Eldrich E. Tromp, Vinod Subramaniam, Jurriaan Huskens, and Pascal Jonkheijm

Journal

JACS

Publication Date

02/04/2013

Abstract

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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Date added: 2013-03-12 13:40:57 | Last time updated: 2013-03-12 12:40:57 | Viewed: 1193 times

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