Trifunctional Click Chemistry Probes that incorporate a ligation handle, a biotin and a fluorophore have become a popular tool for for tandem labeling of proteins and subsequent detection or enrichment. However, due to the strong interaction between biotin and streptavidin harsh conditions are necessary for the elution of enriched proteins. This usually leads to contamination of the sample with non-specifically bound proteins and endogenously biotinlytated proteins, which complicates target identification.Desthiobiotin Trifunctional Click Chemistry Probes overcome this major drawback of the streptavidin-biotin affinity purification. These probes contain a biotin moiety linked to a “clickable” group, and fluorescent dye (TAMRA). Desthiobiotin is a single-ring, sulfur-free analog of biotin that binds to streptavidin with nearly equal specificity but less affinity than biotin (Kd = 1011 vs. 1015M, respectively). Consequently, desthiobiotinylated proteins can be eluted readily and specifically from streptavidin affinity resin using mild conditions based on competitive displacement with free biotin. For pull-down experiments with biological samples, this soft-release characteristic of desthiobiotin also helps to minimize co-purification of endogenous biotinylated molecules, which remain bound to streptavidin upon elution of the target protein complexes with free biotin.Another very important advantage of dual label probes over regular biotin probes is built-in control. Each step of enrichment process can easily followed either by UV-Vis (550 nm) or by more sensitive fluorescence spectroscopy. After elution form streptavidin beads target proteins containing TAMRA label can be easily distinguished from non-specifically bound proteins and endogenously biotinlytated proteins.This new dual label probe should be useful to expand the new applications of standard biotin–streptavidin technology.labeling reagent capable of covalently attaching to a terminal alkyne at one end. Unlike typical fluorescent labeling reagent, Cy5-Biotin-Azide also incorporates a biotin group. Together these features allow for both visualization and affinity purification of alkyne-tagged molecules.
