GlycoLink Immobilization Kits and Resin

Thermo Scientific GlycoLink Immobilization Kits provide affinity columns and reagents to immobilize glycoproteins through oxidized sugar groups for use in affinity-purification applications or studies of protein-protein interactions.

The GlycoLink Kits use hydrazide-activated resin and a special catalyst to prepare affinity columns with polyclonal antibodies, which typically have abundant carbohydrates (glycosylation) on their Fc portions. Any glycosylated proteins (even monoclonal antibodies) that contain periodate-oxidizable carbohydrate groups can be immobilized with these kits to make reusable affinity columns. Antibodies immobilized by this hydrazide immobilization chemistry have unobstructed antigen-binding sites and optimal purification capability. Prepared columns with stable glycoproteins or antibodies can be regenerated and reused at least five times for affinity-purification without significant loss in binding capacity.

Highlights:

• High-capacity – immobilize 1 to 10mg of oxidized antibody or other glycoprotein per milliliter of resin
• Fast and efficient – immobilize in as few as 30 minutes; achieve at least 90% coupling of most glycoproteins in less than 4 hours (efficiency depends on the amount and type of glycosylation)
• Specific immobilization – the hydrazide-activated UltraLink Resin conjugates only to purified glycoproteins whose sugar (e.g., sialic acid) groups have been gently oxidized with periodate
• Stable attachment – resonance structure of the hydrazone bonds are sufficiently stable for multiple affinity purifications with one batch of prepared resin; no stabilization step is required
• Preserves antibody function – immobilizes IgG via the Fc region, thereby keeping both antigen binding sites available for capturing target
• Convenient sizes – choose from two complete 10-column kits: one with 1mL columns and one with 0.1mL columns

Applications:

• Purify antigens using covalently coupled antibodies
• Determine binding partners for a glycoprotein of interest
• Perform large-scale affinity purifications

Includes:

Kits contain GlycoLink Columns; coupling buffer, coupling catalyst and wash buffer; oxidizing reagent; desalting columns, PBS buffer pack; and column accessories

Product Details:

Chemistry of glycoprotein immobilization with Thermo Scientific GlycoLink Resin. Glycoproteins with oxidized sugars contain aldehyde groups which will conjugate to the hydrazide-activated beaded resin support. Learn more about carbonyl-reactive immobilization methods and about hydrazide crosslinking chemistry.

The GlycoLink Immobilization Kit method is especially useful for glycoproteins, such as polyclonal antibodies, because it can allow molecules to be attachmed at a domain that will not interfere with active binding sites that are critical for the intended affinity purification. Carbohydrate moieties in glycoproteins contain common sugars with cis-diol groups that are easily oxidized with sodium meta-periodate to yield aldehydes. When incubated with hydrazide resin, these aldehyde groups react with the hydrazide groups on the resin to form stable, covalent bonds. This reaction is enhanced by the Thermo Scientific GlycoLink Coupling Catalyst, increasing coupling efficiency and decreasing incubation times. The entire coupling reaction occurs in 2 to 4 hours in a simple non-amine buffer containing the catalyst. Coupling efficiencies with antibodies and typical glycoproteins are generally greater than 85%, resulting in 1 to 10mg of immobilized protein per milliliter of Thermo Scientific UltraLink Hydrazide Resin.

Improved immobilization and conjugation of glycoproteins – Efficient aldehyde-hydrazide covalent coupling using a catalyst and an optimized buffer.

General References:

1. Abraham, R., et al. (1991). The influence of periodate oxidation on monoclonal antibody avidity and immunoreactivity. J. Immunol. Methods 144(1):77-86.
2. 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.
3. 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.
4. Dirksen, A., Dawson, PE. (2008). Rapid oxime and hydrazone ligations with aromatic aldehydes for biomolecular labeling. Bioconjug. Chem. 19(12):2543-8.
5. Domen, P., et al. (1990). Site-directed immobilization of proteins. J. Chromatogr. 510:293-302.
6. O’Shannessy, D., et al. (1984). A novel procedure for labeling immunoglobulins by conjugation to oligosaccharide moieties. Immunol. Lett. 8:273-7.
7. Zing, Y., et al. (2009). High-efficiency labeling of sialylated glycoproteins on living cells. Nat. Methods 6(3):207-9.

Related Resources:

GlycoLink FAQ
Review of glycosylation and methods to analyze glycoproteins and glycopeptides
Review of Affinity Purification
Review of Covalent Immobilization Methods
Hydrazide Crosslinker Chemistry

Related Products:

Sodium meta-Periodate
GlycoLink Coupling Catalyst
Gentle Ag/Ab Elution Buffer, pH 6.6
Glycoprotein Carbohydrate Estimation Kit
GlycoLink IP Kits