Oriented protein immobilization using covalent and noncovalent chemistry on a thiol-reactive self-reporting surface

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

    Research output: Contribution to JournalArticleAcademicpeer-review

    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.

    Original languageEnglish
    Pages (from-to)3104-11
    Number of pages8
    JournalJournal of the American Chemical Society
    Volume135
    Issue number8
    DOIs
    Publication statusPublished - 27 Feb 2013

    Keywords

    • Proteins
    • Sulfhydryl Compounds
    • Surface Properties
    • Journal Article
    • Research Support, Non-U.S. Gov't

    Fingerprint Dive into the research topics of 'Oriented protein immobilization using covalent and noncovalent chemistry on a thiol-reactive self-reporting surface'. Together they form a unique fingerprint.

    Cite this