Thermo Scientific EZ-Link Phosphine-PEG4-Desthiobiotin is a pegylated reagent whose biotin-like group is elutable from streptavidin, activated for chemoselective ligation and purification of azide targets tagged by metabolic labeling.
When used in combination with azide labeling strategies, Phosphine-PEG4-Desthiobiotin enables affinity purification of protein interactions and post-translational modifications using streptavidin resins. The phosphine moiety selectively crosslinks via the Staudinger reaction mechanism to molecules that have been tagged or metabolically labeled with bioorthogonal azide groups. The desthiobiotin tag binds to streptavidin and other biotin-binding proteins with high specificity yet readily elutes with mild conditions (i.e., by competitive displacement with regular, free biotin). As such, this reagent is a useful alternative to Phosphine-PEG3-Biotin (Part No. 88901) for avidin-biotin techniques in which nondenaturing elution of the labeled proteins is desired. The No-Weigh Format (5 x 1mg resealable vials) eliminates the difficulties associated with weighing small quantities of reagent and maximizes protection of unused reagent from degradation.
- Desthiobiotin – analog of biotin allows easy elution from streptavidin, an ideal feature for affinity purification applications
- Chemoselective – the phosphine reactive group is specific in biological samples for azide-tagged molecules, ensuring specific capture and purification of labeled targets
- Compatible – Staudinger reaction chemistry proceeds efficiently in simple buffer conditions; requires no accessory reagents such as copper or reducing agents
- PEG spacer – polyethylene glycol spacer arm helps maintain solubility of labeled molecules and decreases steric hindrance for affinity-binding to avidin, streptavidin or NeutrAvidin Protein
- Soluble – easily dissolves in water-miscible solvents (e.g., DMSO) for subsequent dilution in aqueous reaction mixtures with cell lysates and other biological samples
Properties of Phosphine-PEG4-Desthiobiotin. We manufacture our reagents to ensure the highest possible overall product integrity, consistency and performance for the intended research applications.
|Spacer arm length
|Mass added to target
Desthiobiotin vs. Biotin:
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.
Labeling with Phosphine Reagents:
Azide groups can be introduced into proteins or other cellular targets through in vivo labeling with azide-derivatives of naturally occurring metabolic building blocks. Because phosphines and azides are foreign and invisible to biological systems (bioorthogonal), they comprise a chemoselective (mutually specific) ligation pair for labeling and conjugation.
|Overview of Staudinger ligation (azide-phosphine conjugation). Phosphine-activated compounds conjugate with high specificity to azide-tagged molecules, resulting in stable covalent attachment of "A" and "B" molecules. For additional information, see our review of Staudinger ligation reaction chemistry.
- 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)
Azide-Phosphine Reagent Selection Guide
All Biotinylation Reagents
Avidin, Streptavidin, NeutrAvidin Affinity Resins