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.

Oriented Protein Nanoarrays on Block Copolymer Template

“…Moreover, such site-specifi c immobilization can be applied not only to proteins in native states but also to those under denaturing conditions. [39][40][41][42] Thus, when applied to protein nanoarray technology, the IDA/Cu 2+ /poly-His linking chemistry does not need to rigorously purify the protein samples. By combining His-tagged fusion protein and Cu 2+ -functionalized PS spots on PS 60 -b -PHEMA 150 nanopatterns, the method described here demonstrates a simple and effective means to fabricate oriented protein nanoarrays.…”

Polymer‐ and Ionic Liquid‐Containing Palladium: Recoverable Soluble Cross‐Coupling Catalysts

“…A 40 m M NiCl 2 solution was flown through the device for 30 min to coordinate Ni 2+ ions with the NTA‐groups, 100 μL min −1 stop‐flow. After washing the device with PBS, 1 μ M of hexahistidine‐tagged TagRFP, provided by S. Sankaran and Dr. D. Wasserberg,47 in PBS was added at 10 μL min −1 for 30 min. After a final wash with PBS at 10 μL min −1 the device was imaged with confocal microscopy.…”

Selective and reversible photochemical derivatization of cysteine residues in peptides and proteins

“…6 A unique cysteine residue can, however, be readily introduced by site directed mutagenesis providing opportunities for installing novel functionalities. 7,8 Approaches commonly employed for cysteine derivatization include alkylation with bromo- and iodoacetyl derivatives, mixed disulfide formation, or Michael additions to substituted maleimides. 7,9 Some of these method allows for the subsequent removal of the installed tag under different conditions.…”