Diazo Biotin Azide
Diazo Biotin Azide is an azide-activated, sodium dithionite (Na2S2O4) cleavable biotin probe that allows for efficient release of the captured biomolecules from streptavidin. This reagent contains a biotin moiety linked to an azide group through a spacer arm containing an azo group that can be cleaved with 25 mM sodium dithionite solution.
Extraordinary strength of the streptavidin-biotin interaction allows for efficient capturing of even highly dilute targets; however, it makes recovery of proteins from affinity resins challenging. Conventional methods to elute biotinylated proteins from immobilized avidin include the following:
- denaturation of streptavidin by boiling the resin in a denaturing buffer that may include high concentrations of chaotropic salts
- trypsin digestion of proteins while they are bound to the resin, or
- elution of proteins with excess free biotin.
These protocols can co-elute contaminant proteins by releasing nonspecifically bound proteins and/or naturally biotinylated proteins concurrently with labeled proteins. In addition, some of these methods can cause elution of high levels of resin-based peptides along with the proteins of interest, resulting in further sample contamination.
Diazo Biotin-Azide probes eliminate a major limitation of the streptavidin-biotin affinity purification. This reagent contains a biotin moiety linked to an azide moiety through a spacer arm containing a cleavable linker. Captured biomolecules can be efficiently released under mild conditions (25 mM sodium dithionite) and the small (178.19 Da) molecular fragment left on the labeled protein following cleavage. These features make the Diazo probe especially attractive for use in biomolecular labeling and proteomic studies.
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