The Thermo Scientific GlycoLink Coupling Catalyst decreases reaction times and increases aldehyde-hydrazide coupling efficiency, resulting in greater than 90% coupling of glycoproteins in 4 hours.
The GlycoLink Coupling Catalyst is a two component kit composed of aniline and sodium acetate buffer. The catalyst accelerates the coupling rate of hydrazide and alkoxyamine moieties with reactive aldehydes that have been generated by periodate-oxidation of cis-diols in glycoproteins. The GlycoLink Coupling Catalyst increases the coupling efficiency of these reactions, decreasing the molar excess amounts of labeling reagents that are required for conjugation reactions. The catalyst is also useful for increasing the binding efficiency of oxidized glycoproteins or other carbohydrate-containing molecules to hydrazide resins.
- Optimized – catalyzes aldehyde-hydrazide and aldehyde-alkoxyamine conjugation reaction
- Efficient – maximizes coupling efficiency and decreases required coupling reaction incubation time
- Gentle – does not interfere with protein function and is easily removed by desalting or dialysis
- Labeling aldehyde-containing molecules with alkoxyamine-biotins
- Labeling aldehyde-containing molecules with hydrazide-biotins
- Increase coupling efficiency of aldehyde-containing molecules to hydrazide resin
Producing reactive aldehydes via sodium meta-periodate oxidation of cis-diols in glycoproteins is a useful technique for variety of protein labeling and detection methods. The GlycoLink Coupling Catalyst (aniline) increases the efficiency of these reactions by acting as a nucleophilic catalyst that accelerates bond formation through a Schiff base intermediate. The catalyst also reduces the amount (molar excess) of hydrazide or alkoxyamine required in labeling reactions, thereby eliminating the need for DMSO to solubilize high concentrations of labeling reagent.
The Thermo Scientific GlycoLink Coupling Catalyst increases binding efficiency of glycoproteins to hydrazide resin. Human IgG was oxidized with 10mM sodium meta-periodate for 30 minutes. The oxidized IgG (10mg) was diluted to 5mg/mL either with GlycoLink Coupling Buffer alone or with GlycoLink Coupling Catalyst and coupled to 1mL of hydrazide resin. Results are the average of three separate conjugations for each condition after incubating for 4 hours.
- Abraham, R., et al. (1991). The influence of periodate oxidation on monoclonal antibody avidity and immunoreactivity. J. Immunol. Methods 144(1):77-86.
- Byeon, J.Y., et al. (2010). Efficient bioconjugation of protein capture agents to biosensor surfaces using aniline-catalyzed hydrazone ligation. Langmuir 26(19):15430-5.
- Dirksen, A., et al. (2006). Nucleophilic catalysis of hydrazone formation and transimination: implications for dynamic covalent chemistry. J. Am. Chem. Soc. 128(49):15602-3.
- Dirksen, A., Dawson, PE. (2008). Rapid oxime and hydrazone ligations with aromatic aldehydes for biomolecular labeling. Bioconjug. Chem. 19(12):2543-8.
- Domen, P., et al. (1990). Site-directed immobilization of proteins. J. Chromatogr. 510:293-302.
- O’Shannessy, D., et al. (1984). A novel procedure for labeling immunoglobulins by conjugation to oligosaccharide moieties. Immunol. Lett. 8:273-7.
- Zing, Y., et al. (2009). High-efficiency labeling of sialylated glycoproteins on living cells. Nat. Methods 6(3):207-9.
Review of glycosylation and methods to analyze glycoproteins and glycopeptides
Review of Affinity Purification
Review of Covalent Immobilization Methods
Hydrazide Crosslinker Chemistry
GlycoLink Immobilization Kits and Resins
GlycoLink Immunoprecipitation Kit
Hydrazide- and Alkoxyamine-activated Biotinylation Reagents