Thermo Scientific EZ-Link Alkoxyamine-PEG4-SS-PEG4-Biotin is a biotinylation reagent containing a long polyethylene glycol (PEG) spacer and cleavable disulfide bond for reversibly biotinylating at carbohydrate groups that have been oxidized to aldehydes.

Alkoxyamine-activated biotinylation reagents such as this one are useful for biotin-labeling glycoproteins and other molecules that have oxidizable polysaccharides groups. EZ-Link Alkoxyamine-PEG-Biotin reagents contain a multi-functional extended spacer arm that is a flexiblenon-immunogenic hydrophilic polyethylene glycol (PEG) which imparts water solubility to labeled molecules. Consequently antibodies labeled with biotin alkoxyamine reagents exhibit less aggregation when stored in solution compared to antibodies labeled with reagents having only hydrocarbon spacers. Alkoxyamine-PEG4-SS-PEG4-Biotin has a disulfide bond in the spacer and two PEG chains. The disulfide bond enables cleavage of the biotin group from the labeled proteins which is especially useful when affinity purifying biotinylated proteins using immobilized avidin streptavidin or Thermo Scientific NeutrAvidin Protein.

Chemical stucture of Alkoxyamine-PEG4-SS-PEG4-Biotin.

• Biotin – label macromolecules for detection or affinity purification with avidin and streptavidin
• Alkoxyamine-activated – reacts with aldehyde groups (e.g. oxidized sugars in glycoproteins) to attach biotin by stable oxime bonds
• Polyethylene glycol – PEG groups are flexible non-immunogenic hydrophilic and improve the solubility of labeled antibodies
• Long spacer arm – the reach between the point of attachment and the biotin affinity tag is nearly 57 angstroms minimizing steric hindrance for binding
• Cleavable – disulfide bond in middle of spacer arm can be cleaved with reducing agents (e.g. DTT) to release the molecule from the affinity-bound biotin goup

• Chemical formula: C34H64N6O12S3
• Molecular weight: 845.10
• Spacer arm: 56.6 angstroms
• Net mass added to target: 827.08
• Form: viscous semi-solid
• Solubility: dissolve in DMSO to make 250mM stock

The alkoxyamine group reacts to carbonyls (aldehydes and ketones) resulting in an oxime linkage. Sialic acid is a common sugar component of protein polysaccharides and is easily oxidized with 1mM sodium meta-periodate (NaIO4). Other sugars are effectively oxidized with 5-10mM sodium meta-periodate. With proteins oxidation of sugar moieties enables directed labeling away from polypeptide domains that are important for protein function. Because most polyclonal antibodies are glycosylated in regions other than the antigen-binding sites labeling is achieed without adversely affecting antibody function in immunoassays. Note that monoclonal antibodies may be deficient in glycosylation.

Alkoxyamine reagents also can be reacted with carboxyl groups using the carbodiimide EDC (Part No. 22980). EDC activates carboxyl groups to bind to the –NH2 group of the alkoxyamine forming an amide linkage. Using EDC may result in polymerization of the peptide or protein if the molecule has both carboxyls and primary amines on its surface; however decreasing the EDC amount or increasing the amount of the biotin reagent in the reaction can minimize polymerization.

1. Dirksen A. et al. (2006). Nucleophilic catalysis of oxime ligation. Angew Chem Int Ed 45:7581-4.
2. Dirksen A. and Dawson P. (2008). Rapid oxime and hydrazone ligations with aromatic aldehydes for biomolecular labeling. Bioconjugate Chem 19:2543-8.
3. Lempens E. et al. (209). Efficient and chemoselective surface immobilization of proteins by using aniline-catalyzed oxime chemistry. Chembiochem 10:658-62.
4. Mahal L.K. et al. (1997). Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 276:1125-8.
5. Zeng Y. et al. (2009). High efficiency labeling of glycoproteins on living cells. Nat Methods 6:207-9.

Guide to PEGylation – links to different kinds of polyethylene glycol reagents

Alkoxyamine-PEG4-Biotin
Alkoxyamine-PEG12-Biotin
PEGylation Reagents