GlycoLink™ Immobilization Kit - FAQs

The stability of the immobilized protein and the type of elution buffer used determines how many times a column can be reused. Typically, the columns can be reused at least five times without significant loss in performance.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Yes. Chicken antibodies have a total of 4 N-linked glycosylation sites and will couple to the resin.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

The resin will couple any antibody that is adequately glycosylated. Although monoclonal antibodies are typically under-glycosylated, many have been effectively immobilized to this resin. To determine if a monoclonal antibody is glycosylated use the Thermo Scientific Glycoprotein Carbohydrate Estimation Kit (Cat. No. 23260).

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

If too much sodium meta-periodate is used or the sample is incubated for too long the oxidation can cause the protein to breakdown or favor an amine linkage to the resin. Furthermore, if used in excess, methionine residues might become oxidized. Typically, using 10 mM sodium meta-periodate for 30 minutes will oxidize only the carbohydrates and does not affect protein function.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

The antibody or other glycoprotein is first diluted or desalted into the coupling buffer and transferred to a vial containing sodium meta-periodate at a final concentration of 10 mM (1 mM is sufficient for sialic acid residues). The mixture is incubated for 30 minutes at room temperature in the dark. Sodium meta-periodate concentration can be increased to 25mM if oxidation is not sufficient at 10 mM. Care must be taken not to over-oxidize the glycoprotein.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Each glycoprotein will result in a different binding capacity. Binding capacity is dependent on the amount of glycosylation and the type of sugars present. Proteins with mannose and galactose will bind at a higher capacity than proteins with sugars that are not as readily oxidized. Binding capacity of a specific glycoprotein must be determined empirically. The hydrazide-activated UltraLink Resin can bind greater than 15 mg of ovalbumin per milliliter of resin. (Ovalbumin is greater than 3% glycosylated by weight and has a high mannose content.) As a guideline, up to 10 mg of rabbit IgG polyclonal antibody can be coupled to 1 mL of the hydrazide-activated UltraLink Resin.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

The hydrazides on the resin couple to oxidized sugar groups on glycoproteins and other carbohydrate-containing molecules. Sodium meta-periodate must be used to oxidize the cis-diols of sugar groups on glycoproteins to generate the required reactive aldehyde moieties. These aldehydes form stable, covalent hydrazone linkages with the hydrazides on the resin. In the absence of aldehydes, ketone groups also can be targeted for immobilization.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

The GlycoLink Kits (Cat. No.s 88941, 88942) include aniline as a catalyst and optimal pH buffers, which can couple more than twice the amount of protein per milliliter of resin compared to the resin in the CarboLink Kits. Also, GlycoLink Kits use hydrazide-activated UltraLink Resin (Cat. No. 53149, 10 mL) instead of hydrazide-activated agarose resin. Furthermore, we have added kit formats for small-scale coupling (Cat. No. 88942) and immunoprecipitation.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

GlycoLink gel is hydrazide-activated crosslinked beaded agarose, and it is useful for coupling glycoproteins via aldehydes formed from their sugars by sodium meta-periodate oxidation. Reaction of aldehydes with hydrazide- activated resin is catalyzed by aniline resulting in >90% coupling in 4 hours or less. CarboxyLink gel on the other hand is crosslinked beaded agarose activated with diaminodipropylamine (DADPA) and is useful for immobilizing carboxyl- containing biomolecules after EDC activation. Actually, both resins can be used with EDC to couple ligands via carboxylic acids.

Note: Both immobilization chemistries are available on UltraLink Support as UltraLink Hydrazide and UltraLink DADPA respectively.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Yes, Chicken IgY can be immobilized using GlycoLink Gel, due to the high level of glycosylation of the IgY.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

No. Monoclonal antibodies are typically under-glycosylated, and coupling efficiency to CarboLink Gel may be low. However, many monoclonal antibodies can be immobilized using GlycoLink Gel. Try the Pierce Glycoprotein Carbohydrate Estimation Kit (Cat. No. 23260) to determine if your monoclonal antibody is glycosylated.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

The benefits include high-capacity immobilization of 1-10 mg of oxidized antibody or other glycoprotein per milliliter of resin. Immobilization is fast (in as few as 30 mins) and can achieve at least 90% coupling of most glycoproteins in less than 4 hours. The hydrazide-activated UltraLink resin conjugates only to purified glycoproteins whose sugar groups have been gently oxidized with periodate.

No stabilization step is required when using this resin, and antibody function is preserved as IgGs are immobilized via the Fc region, keeping both antigen binding sites available for capturing targets.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.