Click chemistry is the detection method of choice for samples that would be compromised by direct labeling or antibody-based secondary detection techniques. The click label is small enough to penetrate complex samples easily, and the selectivity and stability of the click reaction provide high sensitivity and low background signal. This gentle sample treatment together with the biocompatible Click-iT Plus reaction means that detection can be multiplexed with expressed proteins such as GFP, protein labels such as R-PE, and a wide range of organic fluorophores.
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Click-iT technology overview
Click-iT reactions use azides and alkynes as specific binding moieties in a two-step procedure that first labels and then subsequently detects your molecule of interest. Azides and alkynes can be used interchangeably in “click” reactions, so either one can serve as the tagged substrate or be labeled for the detection step.
Azides and alkynes are extremely small, inert, and stable moieties that can be incorporated into biological molecules. They are small enough that nucleotides, sugars, or amino acids can be tagged and still continue to function in the cell and incorporate normally into nucleic acids, complex polysaccharides, or proteins.
Small molecules permit:
- Cellular loading with minimal disruption
- Normal incorporation into biomolecules
In a typical Click-iT labeling step, a cell is supplied with the azide or alkyne moiety in a tagged molecule and it is incorporated into a cellular biopolymer.
Azides and alkynes bind together in a “click” reaction with high specificity to form stable, inert products. We’ve developed both molecules with fluorescent labels attached, such as Alexa Fluor dyes, to serve as sensitive detection tools that are small enough to penetrate complex samples.
- Cellular labeling with minimal disruption
- Specific detection with low background signal
If an azide is incorporated into a cellular protein it can be labeled using an alkyne tagged with an Alexa Fluor dye. Conversely, if an alkyne is incorporated into a cellular protein it can be labeled using an azide tagged with an Alexa Fluor dye.
The copper-catalyzed click reaction links an alkyne with an azide to form a stable inert product. The mild reaction conditions of the Click-iT Plus reagent help preserve the activity of fluorescent proteins, so click reactions can be multiplexed with GFP or R-PE in your detection strategy.
Molecular Probes Handbook
BioProbes Journal articles
For Research Use Only. Not for use in diagnostic procedures.