Dr. Brian J. Agnew Profile

Dr. Brian J. Agnew

Senior Scientist at Molecular Probes Inc

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Proceedings Article | 22 February 2008 Paper

Click chemistry for labeling and detection of biomolecules

Brian Agnew, Suzanne Buck, Tamara Nyberg, Jolene Bradford, Scott Clarke, Kyle Gee

Proceedings Volume 6867, 686708 (2008) https://doi.org/10.1117/12.762175

KEYWORDS: Proteins, Chemistry, Luminescence, Flow cytometry, Macromolecules, Ruby, Molecules, Copper, Analog electronics, Multiplexing

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Described herein is an application of the copper(I)-catalyzed Huisgen [3+2] cycloaddition between azides and alkynes, or click chemistry, for the universal two-step detection of biological macromolecules. The first step involves the metabolic incorporation of an azide or alkyne probe into a macromolecule of interest. The second step involves the click chemistry conjugation of the labeled macromolecule with a partner alkyne or azide-reactive fluorescent probe to form a stable triazole ring conjugate. The fluorescently tagged molecules can be subsequently detected by a number of different fluorescent readout platforms including flow cytometry, fluorescence imaging, and 1-D/2-D gel imaging. We demonstrate application of this technology in two different labeling schemes. First, the labeling of newly synthesized DNA in a novel cell proliferation assay, and second, in the labeling of specific glycoprotein subclasses for biomarker discovery applications. In each case, azide or alkyne probes are introduced metabolically with subsequent detection using click chemistry. Utilization of the cellular enzymatic machinery for high-fidelity target molecule labeling combined with the superior efficiency of click chemistry detection results in highly versatile macromolecular labeling platforms that are unmatched in sensitivity and selectivity.

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