Thermo Scientific™ EZ-Link™ Desthiobiotinylation and Pull-Down Kit

Description

• 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
• Highlights: 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 Product Details: 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.