EZ-Link™ Hydrazide-PEG4-Desthiobiotin, No-Weigh™ Format

Thermo Scientific EZ-Link Hydrazide-PEG4-Desthiobiotin is a pegylated, carbonyl-reactive labeling reagent whose biotin-like group is elutable from streptavidin, enabling its use for cell surface glycoprotein labeling and purification.

Features of EZ-Link Hydrazide-PEG4-Desthiobiotin:

• Desthiobiotin—analog of biotin allows easy elution from streptavidin, an ideal feature for affinity purification applications
• Glycoprotein labeling—tag glycosylated proteins at sialic acid residues to enable high recovery in pull-down assays with streptavidin beads
• Cell surface labeling—tag and isolate cell surface glycoproteins; reagent does not permeate membranes of whole cells
• Aldehyde-reactive—reacts with aldehydes formed by periodate-oxidation of sugar groups
• Hydrazide-activated—perform reactions at pH 4 to 6 in buffers such as sodium acetate
• Pegylated—spacer arm contains a hydrophilic, 4-unit, polyethylene glycol (PEG) group
• Enhances solubility—pegylation imparts water solubility to the biotinylated molecule, helping to prevent aggregation of biotinylated antibodies stored in solution

This desthiobiotin variant of biotin is activated to label periodate-oxidized carbohydrates of macromolecules. The hydrazide group conjugates to carbonyls (aldehydes and ketones), such as those created from sialic acid and other sugar components of glycoprotein polysaccharides upon treatment with 1 to 10 mM sodium periodate (NaIO4). 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 Hydrazide-PEG4-Biotin (Part No. 21360) for avidin-biotin techniques in which nondenaturing elution of the labeled proteins is desired. The hydrophilic, 4-unit polyethylene glycol (PEG) spacer arm enhances solubility and reduces aggregation of labeled proteins stored in solution. The PEG segment also adds length and flexibility to the spacer arm, minimizing steric hindrance involved with binding to avidin molecules. The No-Weigh Format (5 x 1 mg resealable vials) eliminates the difficulties associated with weighing small quantities of reagent and maximizes protection of unused reagent from degradation.

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 Hydrazide Reagents
Hydrazides and alkoxyamines are two types of carbonyl-reactive groups. Hydrazides (–NH-NH2) react specifically with aldehyde groups in slightly acidic conditions to form hydrazone linkages; these can be further reduced to stable secondary amine bonds using sodium cyanoborohydride (Part No. 44892). The reaction is more efficient in the presence of aniline (Part No. 88944). Alternatively, hydrazides can be conjugated to carboxylic acids using EDC carbodiimide chemistry.

Reactive aldehyde groups can be generated in glycoproteins and other polysaccharide compounds by oxidation of constituent sugar diols using sodium periodiate (Part No. 20504). Sialic acid residues are common components of protein glycosylation and are easily converted to aldehydes with 1 mM NaIO4.