Click-iT Labeling and Detection Tools

Standard reactions

Azides and alkynes can be used interchangeably in standard click reactions, so either one can serve as the tagged substrate or be labeled for the detection step. Some biomolecules are sensitive to the amount of copper used to catalyze standard click reactions. In particular, proteins such as GFP and R-PE exhibit reduced fluorescence and nucleic acids can be denatured by a high-copper environment.

Copper sensitivity

Biomolecules exhibit varying sensitivity to copper concentration. The high copper concentration used to catalyze traditional click reactions maintains the fluorescence of APC, but not GFP. At medium copper concentrations, GFP and APC fluorescence is maintained, but phalloidin binding is lost (Table 1). With Click-iT Plus reagents, the copper concentration can be optimized to preserve the signal in your fluorescent proteins and also drive the EdU click reaction.

Table 1. The effects of copper on fluorescence and phalloidin binding.

Picolyl azides for low-copper reactions

Click-iT Plus reagents enable researchers to perform copper-catalyzed click reactions with compounds that are sensitive to copper, while retaining all of the benefits of the original azide/alkyne click reaction. Each Click-iT Plus picolyl azide–based tool box includes everything required to optimize the copper concentration and protect against undesired copper side reactions with proteins (e.g., GFP, RPE), nucleic acids (e.g., RNA, oligos), and even small molecules (e.g., phalloidin). 

Additionally the Click-iT Plus picolyl azides can be used to increase sensitivity or accelerate the reactions by using the original copper-catalyzed reaction conditions.

DIBO alkynes for copper-free reactions

DIBO reagents can be used to perform click reactions with azide-modified targets without the use of heavy metal catalysis. DIBO reactions are ideal for surface labeling of live cells and also minimize damage to fluorescent proteins like GFP or R-PE. For intracellular labeling, the nonspecific binding of DIBO alkynes creates a background signal, and a labeling strategy using picolyl azides is the preferred solution in these instances.

For added convenience, we offer Click-iT Reaction Buffer Kits optimized for either proteins or cell samples labeled with an azide- or alkyne-tagged biomolecule.

• The Click-iT Cell Reaction Buffer Kit includes sufficient reagents to perform 50 reactions based on a 0.5 mL reaction volume for subsequent analyses by flow cytometry, fluorescence microscopy, or high-content screening (HCS).
• The Click-iT Protein Reaction Buffer Kit includes everything required to perform the click reaction of proteins for subsequent standard protein biochemical analyses (e.g., western blots, mass spectrometry).