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GlycoLink Immobilization Kit FAQ


Answers to frequently asked questions (FAQs) about GlycoLink Immobilization Kit and Hydrazide UltraLink Resin


 

What is the difference between the GlycoLink Kits and the discontinued CarboLink Kits?
The GlycoLink Kits 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 instead of hydrazide-activated agarose resin. Furthermore, we have added kit formats for small-scale coupling and immunoprecipitation.

How does the Hydrazide UltraLink Resin immobilize molecules?
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.

How much glycoprotein can I couple per 1mL of Hydrazide UltraLink Resin?
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 15mg of ovalbumin per milliliter of resin. (Ovalbumin is > 3% glycosylated by weight and has a high mannose content.) As a guideline, up to 10mg of rabbit IgG polyclonal antibody can be coupled to 1mL of the hydrazide-activated UltraLink Resin.

How do I oxidize a glycoprotein or antibody?
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 10mM (1mM 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 10mM. Care must be taken not to over-oxidize the glycoprotein.

When oxidizing with sodium meta-periodate, is it possible to over-oxidize the antibody?
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 10mM sodium meta-periodate for 30 minutes will oxidize only the carbohydrates and does not affect protein function.

How do I determine the degree of antibody coupled to the resin?
Protein samples can be quantified using the Thermo Scientific Coomassie Plus (Bradford) Protein Assay (Part No. 23236). See our Tech Tip #9: Quantitate Immobilized Protein. Alternatively, coupling can be estimated by measuring the protein concentration before and after coupling. The difference in the two measurements is the amount coupled to the resin. Make sure to factor in any change in sample volume.

Can the Hydrazide UltraLink Resin couple all types of antibodies?
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 (Part No. 23260).

Will Hydrazide UltraLink Resins couple chicken IgY?
Yes. Chicken antibodies have a total of 4 N-linked glycosylation sites and will couple to the resin.

How many times can the same affinity column be used for purification?
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.

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