Learn about desthiobiotin and other elutable or reversible avidin-biotin affinity systems.

Desthiobiotin is a modified form of biotin that binds less tightly to avidin and streptavidin than biotin while still providing excellent specificity in affinity purification methods. Unlike biomolecules that are labeled with biotin, proteins and other targets that are labeled with desthiobiotin can be eluted without harsh, denaturing conditions. This guide describes available desthiobiotin labeling reagents and demonstrates desthiobiotinylation as an alternative to biotinylation for applications requiring gentle elution and recovery of the labeled target. Other reversible avidin-biotin binding methods are also mentioned.

Learn about desthiobiotin and other elutable or reversible avidin-biotin affinity systems.

Learn about desthiobiotin and other elutable or reversible avidin-biotin affinity systems.

Learn about desthiobiotin and other elutable or reversible avidin-biotin affinity systems.

Desthiobiotin vs. biotin

Limitations of biotin for affinity purification

Because of its especially high affinity and binding specificity, the biotin-avidin (or biotin-streptavidin) interaction has been used for several decades as the basis of many laboratory research techniques to label, detect and purify biomolecules. However, one limitation of the biotin-avidin interaction in purification applications is that it is essentially irreversible under physiological conditions. For example, once the biotinylated proteins in a sample are successfully captured by streptavidin agarose or magnetic beads, they cannot be dissociated (eluted and recovered) without using harsh conditions (boiling, extreme pH, or denaturants). These conditions tend to denature the proteins of interest or cause artifacts in downstream applications, such as gel electrophoresis.

Desthiobiotin structure and properties

To overcome this limitation, new reagents and assays based on desthiobiotin, a non-sulfur containing biotin analog, are being more widely developed and utilized. Desthiobiotin binds less tightly to avidin and streptavidin than biotin (Kd = 10^-11 M versus Kd = 10^-15 M, respectively) but nonetheless provides a high level of specificity


Figure 1. Comparison of the chemical structures of biotin and desthiobiotin.

Unlike biotinylated proteins, desthiobiotinylated bait proteins and their interacting partners can be readily and specifically eluted under mild conditions when captured on streptavidin by using a biotin elution buffer. The soft release characteristic of desthiobiotin minimizes the isolation of naturally biotinylated molecules that can interfere with results and also eliminates the use of harsh elution conditions which can disassociate complexes and/or damage the target protein or cell. This technique is ideal when using native or recombinant proteins that are not expressed with a fusion tag and when you want to isolate captured proteins under native conditions such as targeting intact cells or cell surface proteins.


Figure 2. Elution comparison between desthiobiotin and biotin in pull-down experiments using streptavidin magnetic beads. Purified IgG was labeled with equivalent desthiobiotin or biotin reagents, spiked into A431 cell lysate, and then purified using streptavidin magnetic beads. Different fractions from each pull-down experiment were electrophoresed and the IgG was detected by Western blot. S = starting labeled IgG samples; FT = flow-through; E1-E2 = elution fractions (desthiobiotin pull-down eluted with 4mM biotin; biotin pull-down eluted with 0.5% formic acid, 30% acetonitrile); BB1-BB2 = bead boil fractions. As the red arrows indicate, desthiobiotinylated IgG elutes easily by gentle, competitive displacement with 4mM biotin, while biotinylated IgG elutes effectively only upon boiling.

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Menu of desthiobiotin products

EZ-Link Desthiobiotin Reagents

ProductsActivationLabeling application   
NHS-DesthiobiotinNHSPrimary amines
(lysines) in proteins
Amine-reactive chemistry
Primary amines
(lysines) in proteins
Amine-reactive chemistry
Amine-PEG4-DesthiobiotinAmineCarboxyl groups
(via EDC crosslinker)
Carboxyl-reactive chemistry
Hydrazide-PEG4-DesthiobiotinHydrazideCarbonyl groups
(oxidized carbohydrates in glycoproteins)
Carbonyl-reactive chemistry
Phosphine-PEG4-DethiobiotinPhosphineAzide groups
(created by metabolic labeling)
Staudinger ligation chemistry

Menu of assays and kits that use desthiobiotin

ProductsDesthiobiotin reagent   
Desthiobiotinylation and Pull-Down KitSulfo-NHS-LC-Desthiobiotin   
Label a purified protein and use it to affinity purify interaction complexes in which it participates
RNA 3' End Desthiobiotinylation KitDesthiobiotinylated Cytidine Bisphosphate   
Enzymatically label an RNA oligo for use in Magnetic RNA-Protein Pull-Down Kit
Active Serine Hydrolase ProbesDesthiobiotin-fluorophosphonate   
Covalently label the active site of serine hydrolases to enable their selective enrichment
GTPase Enrichment KitDesthiobiotin-GTP   
Covalently label the active site of GTPases and the GTPase subunits of G-protein coupled receptors to enable their selective enrichment
Kinase Enrichment KitsDesthiobiotin-ATP;
Covalently label the active site of ATPases, including chaperones and metabolic enzymes, to enable their selective enrichment

Other reversible avidin-biotin strategies

Modified avidin

Elutable avidin-biotin systems have been created by modifying the avidin protein so that it has lower affinity towards biotin. Monomeric Avidin Agarose, which consists of avidin that has been immobilized and maintained primarily in monomeric form, and CaptAvidin™ Protein, which has a nitrated tyrosine group in its biotin-binding site, are examples of this sort of strategy.

Cleavable biotin reagents

Another approach is to use biotinylation reagents that contain a disulfide bond (-S-S-) or other easily cleavable group in their spacer arms; Sulfo-NHS-SS-Biotin is an example of such a reagent. A protein labeled with this reagent can be recovered from its bound state with streptavidin resin by reduction (cleavage) of the disulfide bond in spacer arm between the biotin and protein; in this case, the biotin group is left bound to the streptavidin and the protein is recovered without a biotin label. However, native disulfide bonds in the protein of interest also may be cleaved with this method. Sulfo-NHS-SS-Biotin is used as the basis for isolation of cell surface proteins in the Pierce Cell Surface Isolation Kit.

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Iminobiotin is a cyclic guanidino analog of biotin that exhibits considerably weaker binding to avidin. This form of biotin has limited utility in most practical applications. Its primary use is as an affinity resin for purifying functional streptavidin or avidin proteins.


  1. Hirsch JD, et al. (2002). Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal Biochem. 308:343-57.
  2. Hofmann K, et al. (1984). Synthesis of biotinylated and dethiobiotinylated insulins. Biochem. 23:2547-53.
  3. Hofmann K, et al. (1982). Avidin binding of carboxyl-substituted bihotin and analogues. Biochem. 21:978-84.