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. Cleavable biotin probes can help overcome this limitation of the streptavidin-biotin affinity purification. These reagents contain a biotin group linked to clickable moiety through a spacer arm containing a cleavable linker. Captured biomolecules can be efficiently released under mild conditions and the small molecular fragment left on the labeled protein following cleavage.
Figure 1. Diazo Biotin Probe.
The first application of diazobenzene derivatives (Diazo Biotin Probes) as a chemoselective cleavable linker system was reported by Steven Verhelst in 2007. It was demonstrated that this linker can be incorporated into smallmolecule probes and allow for the isolation of specific protein targets by affinity purification. Since the initial publication a number of reports demonstrated that cleavable Diazo Biotin Probes is valuable tool in chemical biology. The major drawback of the first generation diazobenzene-based cleavable probes is low cleavage efficiency. Diazobenzene reductions require multiple washings and high concentrations of the reducing agent, often requiring up to 3 treatment with cleavage cocktail (25 mM sodium dithionate) for the optimal cleavage conditions .
The second generation of diazobenzene-based cleavable probes, termed HAZA Biotin Probes, is structurally optimized azobenzene scaffold with significantly improved cleavage kinetics and efficiency (Fugure 2).
Figure 2. Schematic representation of protein release captured with HAZA Biotin Azide probe.
The efficiency of selective cleavage of HAZA linker is at least 100-fold higher compared to Diazo linker . Proteins captured onto streptavidin beads with HAZA biotin probe can be eluded with 5 mM sodium dithionite in 5 minutes with more than 95% efficiency . In comparison, elution of proteins captured with diazo linker requires 100 mM sodium dithionite or several elutions with 25 mM dithionite .
Figure 3. Reduction kinetics of HAZA–biotin with 1 mM sodium dithionite in two different medias.
The cleavage of the HAZA linker (monitored by UV spectroscopy at 460 nm) showed a half-life of 1 s and a total cleavage time of 10 s with 1 mM dithionite. Similar results were obtained in biological media (Figure 3). This result demonstrates the potency of HAZA biotin probes as a highly efficient cleavable linker.
Combination of very efficient elution and mild cleavage conditions makes HAZA biotin probes especially attractive for use in proteomics and other biomolecular labeling studies.
|Description||Product #||Pkg. Size||Price|
|HAZA Biotin Azide||1435 – 1||1 mg||$89.00|
|1435 – 5||5 mg||$239.00|
|1435 – 25||25 mg||$679.00|
|HAZA Biotin Alkyne||1436 – 1||1 mg||$89.00|
|1436 – 5||5 mg||$235.00|
|1436 – 25||25 mg||$679.00|
|Click-&-Go™ HAZA Protein Enrichment Kit, for enrichment azide-modified proteins||1446||1 kit||$349.00|
|Click-&-Go™ HAZA Protein Enrichment Kit, for enrichment alkyen-modified proteins||1447||1 kit||$349.00|
1. Leriche G.,et al.(2010). Optimization of the Azobenzene Scaffold for Reductive Cleavage by Dithionite; Development of an Azobenzene Cleavable Linker for Proteomic Applications.Eur. J. Org. Chem.23: 4360–64.
2. Chuch N.C.,et al.(2010). Nondenaturing Chemical Proteomics for Protein Complex Isolation and Identification. ChemBioChem.11: 2359–61.
3. Steven H. L.,et al.(2007). A Mild Chemically Cleavable Linker System for Functional Proteomic Applications. Angew. Chem. Int. Ed.,.21: 1284-6.
4. Ying-Yu Y.,et al.(2011). Identification of lysine acetyltransferase p300 substrates using 4-pentynoyl-coenzyme A and bioorthogonal proteomics. Bioorg. Med. Chem. Lett.,.21: 4976-79.