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Thermo Scientific™ GlycoLink™ Micro Immobilization Kit

Catalog No. PI88942
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PI88942 10 Columns
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Catalog No. PI88942 Supplier Thermo Scientific™ Supplier No. 88942

Description

Immobilize polyclonal antibodies or other glycoproteins, via periodate-oxidized sugars, onto hydrazide-activated polyacrylamide beads for AP, IP or pull-down assays.

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

UltraLink Hydrazide Resin:

  • Ligand: hydrazide
  • Support: Thermo Scientific UltraLink Biosupport
  • Spacer arm: 22 atoms
  • Loading: activated with at least 15μmol hydrazide per milliliter of settled resin
  • Format: 50% resin slurry in 0.02% sodium azide

Includes:

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

Recommended for:

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

Specifications

Capacity (Metric) Immobilizing 50 to 500 μg of GlycoProtein per Column
Description GlycoLink Micro Immobilization Kit
Content And Storage Sufficient For: Immobilizing 50 to 500 μg of glycoprotein per column
• GlycoLink Columns, 0.1 mL, 10 columns
• GlycoLink Coupling Buffer, 100 mL
• Aniline, 0.2 mL
• Wash Buffer, 60 mL
• Sodium meta-periodate, 0.5 g
• Zeba Spin Desalting Columns, 0.5 mL, 10 columns
• PBS Pack (makes 500mL), 1 pack
• Column Accessories, 10 ea

Store at 4°C.
Product Line GlycoLink
Quantity 10 Columns
Type Immobilization Kit
Stationary Phase Hydrazide
Sufficient For 10 Columns
Format Kit, Microcentrifuge, Spin Column
Column Type Affinity, UltraLink™ Resin

Frequently Asked Questions (FAQs)

How many times can the same affinity column in the GlycoLink Immobilization Kit 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.
Will Hydrazide UltraLink Resins in the GlycoLink Immobilization kit couple chicken IgY?

Yes. Chicken antibodies have a total of 4 N-linked glycosylation sites and will couple to the resin.
Can the Hydrazide UltraLink Resin in the GlycoLink Immobilization kit 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 (Cat. No. 23260).
When oxidizing with sodium meta-periodate, is it possible to over-oxidize the antibody when using the GlycoLink Immobilization Kit?

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.
How do I oxidize a glycoprotein or antibody when using the GlycoLink Immobilization Kit?

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.
How much glycoprotein can I couple per 1mL of Hydrazide UltraLink Resin when using the GlycoLink Immobilization Kit?

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.

How does the Hydrazide UltraLink Resin immobilize molecules in the GlycoLink Immobilization Kit?

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.

What is the difference between the GlycoLink Kits and the discontinued CarboLink Kits?

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.
What is the difference between GlycoLink and CarboxyLink Gels?

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.
Will Glycolink Gel work with chicken IgY?

Yes, Chicken IgY can be immobilized using GlycoLink Gel, due to the high level of glycosylation of the IgY.
Will GlycoLink Gel work well for conjugating all types of antibodies?

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.
What are the benefits of using a GlycoLink Coupling Resin?

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.

For Research Use Only. Not for use in diagnostic procedures.