Classic click reactions involve a copper-catalyzed azide–alkyne cyclo­addition [1] to label and detect molecules of interest in cells or tissues. A drawback of this approach is that copper ions can harm cells, reduce the fluorescence of fluorophores, and impair protein function. Our new Click-iT® DIBO reagents employ copper-free click chemistry reactions that capture or detect azide-substituted targets, including our metabolic analogs of proteins and many of their posttranslationally modified forms.

DIBO Core Preserves Protein Function and Cell Viability

The key to copper-free click chemistry is the DIBO (dibenzocyclo­octyne) core of the dye-containing derivative [2]. The strain in the eight-membered ring allows the reaction with azide-modified molecules to occur in the absence of catalysts, enabling the study of the surface of live cells, and preventing copper-induced damage of fluorescent proteins such as GFP in fixed and permeabilized cells (Figure 1).

Click-iT® Alexa Fluor® 647 DIBO Alkyne 
Figure 1. Live-cell labeling of surface glycoproteins with Click-iT® Alexa Fluor® 647 DIBO Alkyne. U2OS cells expressing galectin-GFP were treated for 3 days with azide-modified mannosamine (ManNAz), a metabolic glycoprotein labeling reagent for introducing the N-azidoacetyl analog of sialic acid (SiaNAz) into cell-surface glycoproteins. Click-iT® Alexa Fluor® 647 DIBO Alkyne was used to detect SiaNAz moieties (magenta) on the surface of live cells. The galectin-GFP signal (green) is well maintained.

New Products for Efficient, Stable Reactions

Nine new Click-iT® products are available, including DIBO derivatives of Alexa Fluor®, TAMRA™, and biotin labels, and reactive probes capable of modifying amine, cysteine, and carboxy groups.* These tools enable efficient click reactions that do not require extreme temperatures or solvents. The reaction components are biologically inert, and the reaction products are stable, containing an irreversible, covalent bond. The stability of the covalent 1,2,3-triazole linkage between the azide and alkyne reaction partners allows extensive washing to produce high signal-to-noise ratios, an important consideration when detecting intracellular targets in fixed and permeabilized cells. Learn more about our entire line of click-based tools for metabolic and enzymatic analysis of proteins at Click Chemistry Tools for Biological Assays.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.