title: Oriented protein immobilization using covalent and noncovalent chemistry on a thiol-reactive self-reporting surface.
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.
authors: Molecular Nanofabrication Group, MESA+ Institute for Nanotechnology, Department of Science and Technology, University of Twente, 7500 AE, Enschede, Netherlands.
authors: Dorothee
authors: D
authors: Wasserberg
authors: Carlo
authors: C
authors: Nicosia
authors: Eldrich E
authors: EE
authors: Tromp
authors: Vinod
authors: V
authors: Subramaniam
authors: Jurriaan
authors: J
authors: Huskens
authors: Pascal
authors: P
authors: Jonkheijm
mesh_terms: D011506:Proteins / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013499:Surface Properties
chemical_list: D011506:Proteins; D013438:Sulfhydryl Compounds