The efficiency of a copper (Cu(I))-catalyzed Azide-Alkyne click chemistry reaction (CuAAC) strongly depends on the presence of copper ions in the +1 oxidation state (Cu(I)).
Different copper catalyst sources, reduction reagents and Cu(I) stabilizing ligands are available however, for most bioconjugation applications the combination of CuSO4 as copper catalyst source, sodium ascorbate as a reduction reagent and a water-soluble Cu(I) stabilizing ligand such as THPTA[1,2] or BTTAA[3,4] is recommended.
An optimal balance between reaction speed and Cu(I) concentration can be achieved using THPTA or BTTAA in combination with Picolyl-Azide detection reagents (Picolyl-Azides of Biotin or Picolyl-Azides of fluorescent dyes) that contain an additional internal copper chelating moiety.
Presolski et. al. (Download pdf) and Hong et. al. (Download pdf & supplemental information) provide a general protocol for CuAAC reactions that may be used as a starting point for the set up and optimization of individual assays.