DBCO-Sulfo-NHS Ester is water-soluble reagent that enables simple and efficient labeling of antibodies, proteins and any other primary amine-containing macromolecules with DBCO moiety in 100% aqueous buffers.
DBCO-Sulfo-NHS Ester is a reagent of choice for applications that cannot tolerate organic co-solvents or are complicated by their inclusion. Specific labeling of cell surface proteins is another common application for these uniquely water-soluble and membrane impermeable reagents.
|Solubility||Water, DMSO, DMF|
|Appearance||White to slightly grey crystalline|
|Storage Conditions||-20°C. Desiccate|
|Shipping conditions||Ambient temperature|
Protein Labeling Calculator
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Volume of DMSO required to make a stock solution (µL)
Volume of protein to be labeled (µL)
Volume of biotin stock solution to add (µL)
Percent DMSO in Reaction Mixture (%)
Caution - Volume of DSMO solvent added to protein must always be <5% total volume of reaction
Important Product Information
- DBCO-sulfo-NHS ester is moisture-sensitive. To avoid moisture condensation onto the product always let vial come to room temperature before opening; be careful to limit exposure to moisture and restore under an inert atmosphere. The NHS-ester moiety readily hydrolyzes and becomes non-reactive; therefore, prepare stock solutions immediately before use. Stock solutions in anhydrous solvents can be kept for several days (freeze when not in use).
- Hydrolysis of DBCO-sulfo-NHS ester is a competing reaction. Conjugation with primary amines of proteins/peptides (i.e., acylation) is favored at near neutral pH (6-9) and with concentrated protein solutions. For conjugation, use non-amine-containing buffers at pH 7-9 such as PBS (20 mM sodium phosphate, 150 mM sodium chloride, pH 7.4); 20 mM HEPES; 100 mM carbonate/biocarbonate; or 50 mM borate buffer.
- Do not use buffers that contain primary amines, (e.g., Tris, glycine).
- Avoid buffers that contain azides, which can react with DBCO.
- Dissolve DBCO-Sulfo-NHS ester in a aqueous buffer or dry water-miscible organic solvent such as DMSO or DMF before diluting in final reaction buffer.
- Reactions with DBCO and azides are more efficient at high concentrations and temperatures (i.e., 2-37°C). Typical reaction times are less than 4 hours; however, incubating for longer can improve efficiency.
Procedure for Sample Labeling
Additional Materials Required
- Aqueous buffer or water-miscible organic solvent such as dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF)
- Reaction buffer: Phosphate-buffered saline (PBS) or other buffer at pH 5-9
- Quenching buffer: 1 M Tris∙HCl, Ph 8.0
- Spin Desalting Columns
- Prepare proteins in PBS.
- Immediately before use, prepare 10 mM of the DBCO-sulfo-NHS ester in aqueous buffer, DMSO or DMF.
- Add the NHS reagent to the protein sample at a final concentration of 0.5-2 mM. If the protein concentration is ≥ 5 mg/ml, use a 10-fold molar excess of the reagent. For samples < 5 mg/ml, use a 20- to 50-fold molar excess.
- Incubate the reaction at room temperature for 30 minutes or on ice for 2 hours.
- Stop the reaction by adding Quenching Buffer to a final concentration of 50-100 mM Tris.
- Incubate the reaction at room temperature for 5 minutes or on ice for 15 minutes.
- Remove non-reactive reagent by dialysis or desalting.
Copper-free Click Reaction
- Prepare the azide-containing sample in reaction buffer.
- Add DBCO-protein conjugate to azide-containing sample.
Recommendation: Add 1 mole equivalent of limiting reagent to 1.5-3.0 mol equivalents of highest abundance reagent.
- Incubate the reaction at room temperature for 4-12 hour. Incubation at 40C requires 2-12 hours.
- The reaction is now ready for purification.
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2. Guhuan, L., et al. (2015). Hyperbranched Self-Immolative Polymers (hSIPs) for Programmed Payload Delivery and Ultrasensitive Detection. J. Am. Chem. Soc., 137(36), 11645-11655.
3. Sukwon, J., et al. (2013). Facile Strategy for Protein Conjugation with Chitosan-Poly(ethylene glycol) Hybrid Microparticle Platforms via Strain-Promoted Alkyne-Azide Cycloaddition (SPAAC) Reaction. Biomacromolecules, 14(11), 3892-3902.
4. Sukwon, J., et al. (2012). Fabrication of Chitosan-Poly(ethylene glycol) Hybrid Hydrogel Microparticles via Replica Molding and Its Application toward Facile Conjugation of Biomolecules. Langmuir, 28(49), 17061-17070.