The Thermo Scientific EZ-Link Desthiobiotinylation and Pull-Down Kit is a complete set of reagents to biotin-label purified proteins and use them to purify interaction complexes using streptavidin agarose resin with mild elution conditions.
This all-in-one labeling and purification kit uses Sulfo-NHS-LC-Desthiobiotin, an amine-reactive NHS-ester reagent, to effectively tag a purified or mixed protein sample for use as bait in capturing and purifying protein interactions. The desthiobiotin tags of labeled proteins bind to streptavidin with high specificity yet readily elute with very mild conditions (i.e., by competitive displacement with regular, free biotin). The system enables effective yet gentle capture and isolation (pull-down) of specific protein interaction complexes from cell lysates using streptavidin agarose beads. The complete kit includes ready-to-use 1mg vials of desthiobiotinylation reagent, desalting columns to process labeled protein, and high-capacity streptavidin agarose with buffers to perform several microcentrifuge-scale pull-down assay experiments with ease. Identify novel interactions between a known protein (bait) and previously undiscovered target (prey), or confirm interactions among known bait and prey proteins.
- Sulfo-NHS-LC-Desthiobiotin – label proteins with a biotin-like that specifically binds streptavidin but also easily elutes with mild conditions
- Pull-down assay – simple, optimized protocol and reagents to label proteins and recover (purify) intact, functional protein interaction complexes
- Complete kit – includes labeling reagent and desalting columns to prepare bait-proteins, and streptavidin agarose and elution buffer to perform pull-down
|Chemical structure of Sulfo-NHS-LC-Desthiobiotin. This reagent labels antibodies and other proteins and molecules that contain primary amines (e.g., side-chain of lysine). For more information, see our review of NHS Ester Reaction Chemistry.
Desthiobiotin is a single-ring, sulfur-free analog of biotin that binds to streptavidin with nearly equal specificity but less affinity than biotin (1/Kd = 10^11 vs. 10^15M, respectively)[1-2]. Consequently, desthiobiotinylated bait proteins and their interacting partners can be eluted readily and specifically from streptavidin affinity resin using mild conditions based on competitive displacement with free biotin. For pull-down assay 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. The modified avidin-biotin affinity system also eliminates the need to use harsh elution conditions that might disassociate complexes and/or damage the target protein or cell. Desthiobiotin-based techniques are ideal when using native or recombinant proteins that are not expressed with a fusion tag and when isolating captured proteins under native conditions, such as targeting intact cells or cell surface proteins.
Sulfo-NHS-LC-Desthiobiotin is a variant of biotin that is activated as a sulfo-NHS ester with a long-chain (LC) spacer arm to covalently label primary amines (-NH2) of proteins or other molecules with desthiobiotin groups. The No-Weigh Format (1mg resealable vials) eliminates the difficulties associated with weighing small quantities of reagent and maximizes protection of unused reagent from hydrolysis.
- Hirsch, J., et al. (2002). Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Analytical Biochemistry 308: 343-357.
- Hofmann, K., et al. (1982). Avidin binding of carboxyl-substituted biotin and analogues. Biochemistry 21: 978-984.
Review of Biotinylation Methods and Applications
Chemistry of Crosslinking (and Labeling Reagents)
Desthiobiotin Product Selection Guide
All Biotinylation Reagents
Avidin, Streptavidin, NeutrAvidin Affinity Resins