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Fluorescent Tools for Click Chemistry

effective bio-orthogonal labeling with Chromeo™ Dyes

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The principle of click chemistry

Click chemistry constitutes a novel approach for easy, effective labeling and detection of biomolecules in vitro and in vivo. It is based on the use of biologically unique tags as reaction partners. An azide and an alkyne undergo the copper(I)-catalyzed formation of a triazole, which forms a stable covalent bond. This reaction is based on the azide alkyne Huisgen cycloaddition, which was described by Rolf Huisgen in the 1970s. The addition of the Cu(I) catalyst changes the reaction conditions to room temperature and allows the presence of water. Figure 1 shows the reaction scheme of the click-reaction.

Figure 1: Principle of the click reaction between an azide (R1) and alkyne (R2).
Figure 1: The principle of the click reaction between an azide (R1) and alkyne (R2).

Advantages of click chemistry

Using azide or alkyne tags for labeling proteins, nucleic acids, lipids or sugars has the advantage that neither moiety occurs in nature, and neither reacts with other molecules in cellular systems. Because of its high selectivity, bio-orthogonal click-chemistry can be used for detection in complex biological samples: one of the reaction partners is integrated into the molecule of interest, while the second tag containing a fluorescent label is covalently attached by performing the click-reaction. As the reaction is highly specific and proceeds rapidly towards completion, click chemistry can also be used to synthesize large substances.

Bio-orthogonal labeling with fluorescent Chromeo™ Dyes

Chromeo™ Dyes – Chromeo 488, Chromeo 494, Chromeo 546 and Chromeo 642 – exhibit superior fluorescent properties, broad Stokes shifts, stability towards photobleaching and pH, and low cell toxicity. In addition, they are compatible with most excitation sources including diode lasers, LEDs, tungsten lamps and xenon arc lamps. For bio-orthogonal labeling, Chromeo Dyes are offered as click-reactive azide or alkyne.

Figure 2: Click labeling with Chromeo Dye azides
Figure 2: Click labeling with Chromeo Dye azides.

Chromeo™ Azide and Alkyne advantages

  • High fluorescent intensity
  • Photostability – enables multiple exposures and increased exposure time
  • pH insensitive
  • Low background
  • Ready to use in click-reactions

In addition to click chemistry reagents, Chromeo Dyes are offered in a variety of formats like amine reactive NHS-Esters, which have been shown to label amines in peptides, proteins or amino modified DNA. They are available conjugated to biotin or streptavidin, they are part of our easy-to-use Fluorescent Antibody Labeling Kits, and are offered as optimized Fluorescent Secondary Antibody Conjugates for enhanced detection.

HeLa cells stained by alpha Tubulin mAb and Chromeo 546 Fluorescent Secondary Antibody
Figure 3: Chromeo 546 staining in HeLa cells.

HeLa cells were stained with alpha Tubulin mouse mAb (Clone 5-B-1-2) and Chromeo 546 Goat anti-mouse IgG (Catalog No. 15033).