Biotin-PEG11-oxyamine HCl Salt: Properties & Applications

Biotin-PEG11-oxyamine HCl salt is a heterobifunctional biotinylation reagent combining a reactive oxyamine group with a long PEG11 spacer and a biotin affinity handle, enabling stable oxime-bond formation with ketone- or aldehyde-bearing biomolecules.

Product SKU

B595495

Reactive Group

Oxyamine (–ONH₂) reacts selectively with ketones and aldehydes to form hydrolytically stable oxime bonds

Spacer Arm

11-unit polyethylene glycol (PEG11) chain providing hydrophilicity and reduced steric hindrance

Affinity Tag

Biotin; detected or captured via streptavidin/avidin with sub-nanomolar affinity (Kd ≈ 10⁻¹⁵ M)

Salt Form

Hydrochloride (HCl) salt for improved aqueous solubility and storage stability

Typical Reaction pH

pH 4–6 (mildly acidic conditions favor oxime ligation kinetics)

Primary Applications

Biotinylation of glycoproteins, oxidized nucleic acids, ketone-tagged proteins, and carbohydrate-bearing surfaces

What Is Biotin-PEG11-oxyamine HCl Salt?

Biotin-PEG11-oxyamine HCl salt is a heterobifunctional crosslinker composed of three functional domains: (1) a biotin moiety that binds with extremely high affinity to streptavidin or avidin, (2) an 11-unit polyethylene glycol (PEG11) hydrophilic spacer that minimizes non-specific interactions and imparts aqueous solubility, and (3) a free oxyamine (aminooxy, –ONH₂) group presented as its hydrochloride salt to enhance stability and solubility in aqueous buffers.

The oxyamine functional group undergoes chemoselective condensation with carbonyl compounds—specifically aldehydes and ketones—to form an oxime linkage (–CH=N–O–). This reaction is orthogonal to most standard bioconjugation chemistries, does not require metal catalysts under mildly acidic conditions, and yields a covalent bond that is stable across a broad physiological pH range. As a result, this reagent is particularly well-suited for site-specific labeling strategies where preservation of biomolecule function is critical.

Chemical Structure and Key Properties

The linear architecture of Biotin-PEG11-oxyamine HCl salt places the PEG11 spacer between the biotin head group and the reactive oxyamine terminus. The PEG spacer serves multiple roles: it reduces steric clashes between the bulky biotin group and the target biomolecule, improves overall reagent solubility in aqueous media, and diminishes the likelihood of biotin being buried within a hydrophobic protein interior after conjugation—thereby maintaining streptavidin-binding accessibility.

Key physicochemical considerations include:

• Solubility: The HCl salt form and the PEG11 chain together confer good solubility in water and common aqueous buffers such as PBS or acetate buffer. DMSO can be used as a co-solvent for stock solution preparation when needed.
• Reactivity: Oxyamine groups react optimally with aldehydes and ketones at mildly acidic pH (approximately 4–6). Aniline can be used as a nucleophilic catalyst to accelerate oxime ligation at near-neutral pH.
• Stability: The oxime product formed upon reaction is hydrolytically stable under physiological conditions, providing a robust covalent linkage for downstream detection and capture workflows.
• Selectivity: The oxyamine–carbonyl reaction is highly chemoselective; it does not react with amines, thiols, or other functional groups commonly present on biomolecules under standard reaction conditions.

Applications in Bioconjugation and Life Science Research

Biotin-PEG11-oxyamine HCl salt finds broad utility across multiple research areas that require selective, stable biotinylation of carbonyl-containing targets:

Glycoprotein and Glycan Labeling: Cell-surface glycoproteins can be oxidized with periodate (NaIO₄) to generate aldehyde groups on vicinal diol-containing sugar residues. Biotin-PEG11-oxyamine then reacts selectively with these aldehydes, enabling biotinylation of glycoproteins for subsequent streptavidin-based pull-down, ELISA, flow cytometry, or imaging applications.

Metabolic Labeling: Unnatural ketone- or aldehyde-bearing monosaccharides (e.g., N-levulinoyl mannosamine) can be metabolically incorporated into cell-surface glycans. Biotin-PEG11-oxyamine serves as a chemical reporter probe for detecting these unnatural sugars via oxime ligation, supporting studies of glycan biosynthesis and trafficking.

Oxidized Nucleic Acid Biotinylation: Periodate-oxidized 3′-termini of RNA or the ribose moiety of nucleotides can be biotinylated using oxyamine-functionalized reagents, facilitating capture and detection of specific nucleic acid sequences.

Protein Engineering and Site-Specific Labeling: Proteins engineered to contain p-acetylphenylalanine or other carbonyl-bearing unnatural amino acids can be site-specifically biotinylated with Biotin-PEG11-oxyamine, enabling defined stoichiometry and orientation in antibody-drug conjugates, biosensor surfaces, and structural biology studies.

Nanoparticle and Surface Functionalization: Aldehyde- or ketone-functionalized nanoparticles and solid surfaces can be coated with biotin using this reagent, creating streptavidin-compatible platforms for biosensor development and diagnostic assays.

Recommended Protocol and Handling Guidelines

The following general workflow is recommended for oxime ligation using Biotin-PEG11-oxyamine HCl salt. Researchers should optimize specific conditions for their particular target and application.

Stock Solution Preparation: Dissolve the reagent in ultrapure water or an appropriate aqueous buffer (e.g., 100 mM sodium acetate, pH 4.5–5.5) to prepare a concentrated stock solution (e.g., 10–50 mM). Alternatively, dissolve in anhydrous DMSO for compatibility with water-sensitive formulations. Store aliquots at –20 °C under desiccation and avoid repeated freeze–thaw cycles.

Target Preparation: Introduce a carbonyl group on the biomolecule of interest if not already present. For glycoproteins or vicinal-diol-containing targets, treat with 1–10 mM sodium periodate in the dark at 4 °C for 20–30 minutes, then remove excess periodate by desalting or dialysis against the reaction buffer.

Oxime Ligation Reaction: Add Biotin-PEG11-oxyamine HCl salt at a 10–50-fold molar excess relative to the estimated number of carbonyl sites. Conduct the reaction at pH 4–6 (acetate buffer recommended) at room temperature for 1–4 hours, or at 4 °C overnight. Addition of aniline catalyst (e.g., 10 mM final concentration) can accelerate reaction kinetics at higher pH values (pH 6–7).

Purification: Remove unreacted Biotin-PEG11-oxyamine HCl salt by size-exclusion chromatography, dialysis, or spin filtration using an appropriate molecular weight cutoff membrane, depending on the size of the biotinylated target.

Verification: Confirm biotinylation by streptavidin blot (Western), ELISA, or bead-based pull-down assay. Degree of labeling can be estimated by HABA/avidin colorimetric assay where applicable.

Note: Always consult the product datasheet and applicable safety data sheet (SDS) before use. Handle as a potential irritant; use appropriate personal protective equipment.

Storage, Stability, and Quality Specifications

Biotin-PEG11-oxyamine HCl salt should be stored under the conditions specified on the product datasheet. General guidance for long-term stability includes:

• Temperature: Store at –20 °C in a desiccated environment. Protect from moisture and light.
• Form: The solid HCl salt form is more stable than solutions; prepare working solutions fresh or in small aliquots to minimize freeze–thaw degradation.
• Purity: Research-grade material is typically characterized by HPLC and mass spectrometry to confirm identity and purity. Refer to the Certificate of Analysis (CoA) supplied with each lot for specific purity values.
• Shelf life: When stored correctly, the reagent is expected to remain stable for the period indicated on the product label. Periodically verify activity if stored for extended periods.

For procurement and ordering information for SKU B595495, please contact Aladdin Scientific directly or visit the product page.

Frequently asked questions

What types of biomolecules can be biotinylated with Biotin-PEG11-oxyamine HCl salt?

Any biomolecule that presents or can be engineered to present an aldehyde or ketone group is a suitable substrate. Common examples include periodate-oxidized glycoproteins, ketone-tagged recombinant proteins containing unnatural amino acids, oxidized oligonucleotides, and aldehyde- or ketone-functionalized nanoparticles and surfaces.

Why is a PEG11 spacer included in this reagent rather than a shorter linker?

The 11-unit PEG spacer provides substantial hydrophilicity and molecular flexibility between the reactive oxyamine and the biotin group. This reduces steric hindrance during both the oxime ligation reaction and subsequent streptavidin binding, minimizes non-specific surface adsorption due to aggregation, and keeps the biotin moiety accessible for high-affinity streptavidin capture even when the conjugate is bound to a large biomolecule or surface.

What reaction conditions give the best oxime ligation yield?

Oxime ligation between the oxyamine and carbonyl groups proceeds most efficiently at mildly acidic pH, typically between pH 4 and 6, where the carbonyl is more electrophilic and the reaction kinetics are favorable. For applications requiring near-neutral pH (e.g., pH 6–7) to preserve biomolecule integrity, adding aniline as a nucleophilic catalyst at approximately 10 mM can significantly accelerate the reaction rate. Reaction times of 1–4 hours at room temperature or overnight at 4 °C are generally sufficient.

How do I confirm successful biotinylation after the reaction?

Successful biotinylation can be confirmed by several methods: streptavidin-HRP or streptavidin-fluorophore Western blot detects biotinylated protein bands; ELISA with streptavidin-coated plates quantifies the degree of biotinylation; and bead-based pull-down assays using streptavidin-coated magnetic beads can verify functional capture. The HABA/avidin colorimetric assay provides a quantitative estimate of biotin incorporation where the target allows.

Is Biotin-PEG11-oxyamine HCl salt compatible with NHS ester or maleimide coupling chemistry?

The oxyamine group is orthogonal to NHS ester (amine-reactive) and maleimide (thiol-reactive) chemistries under standard conditions, meaning that Biotin-PEG11-oxyamine will not react with lysine amines or cysteine thiols on proteins. This selectivity makes it an excellent complement to NHS ester or maleimide reagents in multi-step or dual-labeling conjugation strategies where different functional groups need to be addressed independently.

References

1. Dirksen A, Dawson PE. Rapid oxime and hydrazone ligations with aromatic aldehydes for biomolecular labeling. Bioconjug Chem. 2008;19(12):2543-2548. doi:10.1021/bc800310p
2. Zeng Y, Ramya TN, Dirksen A, Dawson PE, Bhaumik JK. High-efficiency labeling of sialylated glycoproteins on living cells. Nat Methods. 2009;6(3):207-209. doi:10.1038/nmeth.1297
3. Rashidian M, Dozier JK, Distefano MD. Enzymatic labeling of proteins: techniques and approaches. Bioconjug Chem. 2013;24(8):1277-1294. doi:10.1021/bc400102w
4. Aladdin Scientific. Product datasheet: Biotin-PEG11-oxyamine HCl salt, SKU B595495.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.