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

D. Wasserberg, Carlo Nicosia, E.E. Tromp, Vinod Subramaniam, Jurriaan Huskens, Pascal Jonkheijm

Research output: Contribution to journalArticleAcademicpeer-review

32 Citations (Scopus)

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 languageUndefined
Pages (from-to)3104-3111
Number of pages8
JournalJournal of the American Chemical Society
Volume135
Issue number8
DOIs
Publication statusPublished - 2013

Keywords

  • METIS-301589
  • IR-90132

Cite this

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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.",
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journal = "Journal of the American Chemical Society",
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Oriented Protein Immobilization using Covalent and Noncovalent Chemistry on a Thiol-Reactive Self-Reporting Surface. / Wasserberg, D.; Nicosia, Carlo; Tromp, E.E.; Subramaniam, Vinod; Huskens, Jurriaan; Jonkheijm, Pascal.

In: Journal of the American Chemical Society, Vol. 135, No. 8, 2013, p. 3104-3111.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Oriented Protein Immobilization using Covalent and Noncovalent Chemistry on a Thiol-Reactive Self-Reporting Surface

AU - Wasserberg, D.

AU - Nicosia, Carlo

AU - Tromp, E.E.

AU - Subramaniam, Vinod

AU - Huskens, Jurriaan

AU - Jonkheijm, Pascal

PY - 2013

Y1 - 2013

N2 - 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.

AB - 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.

KW - METIS-301589

KW - IR-90132

U2 - 10.1021/ja3102133

DO - 10.1021/ja3102133

M3 - Article

VL - 135

SP - 3104

EP - 3111

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 8

ER -