Thermo Scientific EZ-Link NHS-Desthiobiotin is a short, membrane permeable, amine-reactive labeling reagent whose biotin-like group is elutable from streptavidin, making it ideal for intracellular protein labeling and purification.
NHS-Desthiobiotin is a variant of biotin that is activated as an NHS ester to covalently label primary amines (-NH2) of proteins or other molecules with desthiobiotin 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 NHS-Biotin (Part No. 20217) for avidin-biotin techniques in which nondenaturing elution of the labeled proteins is desired. This simplest and smallest variety of amine-reactive desthiobiotin reagent is uncharged and cell membrane permeable; therefore, NHS-Desthiobiotin can be used with intact cells for intracellular protein labeling.
- Desthiobiotin – analog of biotin allows easy elution from streptavidin, an ideal feature for affinity purification applications
- Protein labeling – tag antibodies or proteins in purified or mixed samples to enable high recovery in pull-down assays with streptavidin beads
- Membrane-permeable – achieve intracellular labeling with intact cells because the small, uncharged reagent easily diffuses across cell membranes
- Amine-reactive – reacts with primary amines (-NH2), such as the side-chain of lysines (K) or the amino-termini of polypeptides
||Chemical structure of NHS-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.
Properties of NHS-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
||-20°C with desiccant, protect from moisture, use only fresh solutions
||N-hydroxysuccinimide ester, reacts with primary amines at pH 7.0-9.0
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 NHS Ester Reagents:
N-Hydroxysulfosuccinimide (NHS) esters of biotin are the most popular type of biotinylation reagent. NHS-activated biotins react efficiently with primary amino groups (-NH2) in alkaline buffers to form stable amide bonds. Proteins typically have several primary amines that are available as targets for labeling, including the side chain of lysine (K) residues and the N-terminus of each polypeptide. Most sulfo-NHS esters are directly water soluble but not membrane permeable. Plain NHS esters are less soluble in aqueous buffers but are membrane permeable.
- 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