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Thermo Scientific™ CarboxyLink™ Coupling Resin

Catalog No. PI20266
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PI20266 25 mL
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Catalog No. PI20266 Supplier Thermo Scientific™ Supplier No. 20266
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Description

Covalently immobilize peptide antigens and other carboxylate ligands for affinity purification using DADPA-activated agarose beads and EDC crosslinker.

Thermo Scientific™ CarboxyLink Coupling Resins and Kits provide for the covalent immobilization of peptides or other carboxyl-containing (-COOH) molecules to a porous, beaded resin for use in affinity purification procedures.

These activated resins enable immobilization of carboxylate ligands for use in affinity protocols. CarboxyLink Resin is crosslinked beaded agarose (or acrylamide-based Thermo Scientific UltraLink™ Biosupport) that has been activated with diaminodipropylamine (DADPA) to contain long spacer arms, each of which have a primary amine at the end. When incubated with the resin and the carbodiimide crosslinker EDC (included in the CarboxyLink Immobilization Kit), carboxyl-containing molecules become permanently attached to the support by stable amide bonds. Once a molecule is coupled, the prepared affinity column can be used multiple times in typical protein affinity purification procedures. CarboxyLink Coupling Resins also can be used to immobilize other kinds of molecules using alternative amine-reactive crosslinking chemistries.

Highlights:

  • CarboxyLink Coupling Resins – beaded agarose or acrylamide support activated with diaminodipropylamine (DADPA) for amine-to-carboxyl conjugation using EDC crosslinker

  • Efficient peptide immobilization – easily couple 1 to 2mg of peptide per milliliter of settled resin

  • Stable linkage – immobilization results in covalent attachment of carboxyl groups by amide bonds, allowing for multiple rounds of affinity purification with one batch of prepared resin

  • Flexible and gentle coupling conditions – immobilization reaction completed in simple MES or other non-amine and non-carboxyl, near-neutral buffer, with or without organic solvent

  • Ideal for unmodified peptides – immobilizes peptides with high capacity and various orientations without steric hindrance, allowing for effective use in affinity purification of specific antibodies

  • Convenient kits and product sizes – choose 5-column kits with complete sets of buffers, crosslinker and versatile spin/drip columns containing either type of resin (agarose or polyacrylamide) or choose stand-alone resin for other uses

CarboxyLink Coupling Resin (Part No. 20266, 44899):

  • Ligand: diaminodipropylamine (DADPA)
  • Support: crosslinked 4% beaded agarose
  • Spacer arm: 9 atoms
  • Loading: activated with at least 16μmol amine per milliliter of settled resin
  • Format: 50% resin slurry in 0.05% sodium azide

UltraLink DADPA Resin (Part No. 53154):

  • Ligand: diaminodipropylamine (DADPA)
  • Support: Thermo Scientific UltraLink Biosupport
  • Spacer arm: 14 atoms
  • Loading: activated with at least 40μmol amine per milliliter of settled resin
  • Format: 50% resin slurry in 0.02% sodium azide

Includes:

CarboxyLink Resin Columns (DADPA agarose or UltraLink Support), EDC crosslinker, coupling buffer (MES-buffered saline), wash solution, and column accessories

Specifications

Capacity (Metric) 25 mL
Description CarboxyLink Coupling Resin
Content And Storage Store at 4°C.
Form Liquid
Product Line CarboxyLink
Quantity 25 mL
Target Primary Amine
Type Resin
Stationary Phase Amine
Format Bottle
Column Type Affinity, Agarose Resin
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Frequently Asked Questions (FAQs)

What is the difference between the Thermo Scientific CarboLink (now Glycolink) and CarboxyLink Resins?

The CarboxyLink Resin is for immobilizing carboxyl-containing biomolecules after EDC activation. CarboLink Resin is hydrazide-activated and immobilizes glycoproteins. Carbolink has been replaced with glycolink immobilization kits and resins. Glycolink can also be used to immobilize steroids or other molecules containing ketones. Both resins are capable of coupling ligands via carboxylic acids using EDC (Cat. No. 22980). Both immobilization chemistries are available on UltraLink Resin as UltraLink Hydrazide (glycoproteins) and UltraLink DADPA (carboxyl) Resins.
How many purifications can be performed using the same affinity column with CarboxylLink or DADPA UltraLink resin?

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 10 times without significant loss in purification efficiency.

My peptide is not water-soluble; can I use other solvents for conjugation when using the CarboxylLink or DADPA UltraLink resin?

Yes. When coupling water-insoluble peptides or other molecules, use water-miscible solvents such as ethanol, methanol, DMSO or DMF. Dissolve the peptide in 100% of the water-miscible solvent first and then add this solution to the Conjugation Buffer. Organic solvent concentrations up to 50% in the coupling reaction are compatible. When using high concentrations of organic solvent (> 25%), gradually equilibrate the resin into the organic solvent. For example, wash the resin with 2-3 column volumes of each of the following solutions before adding the sample in 50% organic solvent:

- 95% aqueous, 5% organic solvent
- 85% aqueous, 15% organic solvent
- 75% aqueous,25% organic solvent
- 60% aqueous, 40% organic solvent
- 50% aqueous, 50% organic solvent
Reverse the percent organic solvent and re-equilibrate the resin in 100% aqueous if purifying a sample in aqueous buffer.

How do I determine the degree of antibody coupling to the CarboxylLink or DADPA UltraLink resin?

Protein samples can be quantified using Thermo Scientific BCA Protein Assay (Cat. No. 23225) or Coomassie Plus (Bradford) Protein Assay (Cat. No. 23236). Alternatively, coupling can be estimated by measuring the protein concentration in solution 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.
Are the CarboxylLink and DADPA UltraLink Resins available as kits?

CarboxyLink Coupling Resin is available as a kit (Cat. No. 44899), which contains sufficient components to prepare five reusable affinity columns. The resin is also available separately (Cat. No. 20266, 25 mL). DADPA UltraLink is available only as a kit (Cat. No. 53154). The kits do not contain buffers for affinity purification.
What is the difference between CarboxyLink and DADPA UltraLink Resins?

The CarboxyLink Resin is composed of 6% crosslinked beaded agarose. The DADPA UltraLink Resin is composed of an azlactone and acrylamide polymer, which is more durable than agarose and can withstand pressures up to 100 psi, making it suitable for medium-pressure, fast-flow techniques involving large sample volumes.
How do CarboxyLink and DADPA UltraLink Resins immobilize molecules?

These resins are derivatized with diaminodipropylamine (DADPA). DADPA has a terminal primary amine that enables covalent immobilization of peptides or other carboxyl-containing (-COOH) molecules. When incubated with the resin and the carbodiimide crosslinker EDC (included in the CarboxyLink Immobilization Kit), carboxyl-containing molecules become permanently attached to the resin by stable amide bonds. The immobilized molecules can then be used in affinity purification procedures. CarboxyLink/DADPA UltraLink Coupling Resins can also be used to immobilize other kinds of molecules using alternative amine-reactive crosslinking chemistries.
I'm having problems with coupling our molecule onto the gel when using EDC and CarboxyLink Coupling Resin. How does the buffer pH affect the coupling efficiency onto the gel? Is there any other coupling buffer that can be used other than 0.1 M MES buffer?

There are two reactions occurring when a molecule is conjugated to CarboxyLink Resin in the presence of EDC. These reactions may occur simultaneously (at pH 7.0-7.2) or separately (moving from an acidic pH to a slightly alkaline pH). Adding sulfo-NHS to the reaction along with EDC helps increase the yield of the reaction.

The activation reaction of the molecule’s carboxyls with EDC and Sulfo-NHS is most efficient at pH 4.5-7.2; however, the reaction of NHS-activated or sulfo-NHS-activated molecules with primary amines is most efficient at pH 7-8. For best results, perform the first reaction in MES buffer (or other non-amine, non-carboxylate buffer) at pH 4.7-6, then raise the pH to 7.2-7.5 with phosphate buffer (or other non-amine buffer) immediately before reaction to the amine-containing molecule. The reaction should be done with EDC that is freshly prepared. 
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.
What are the benefits of using a CarboxyLink support?

CarboxyLink can be used to immobilize any protein or peptide via the C-terminus or aspartic or glutamic acid residues. Diaminodiproylamine (DADPA) is immobilized onto an agarose support. The carbodiimide crosslinker, EDC facilitates the formation of an amide bond between the carbon of the carboxylic acid and the nitrogen on the terminal amine of DADPA. This support is compatible with 50% organic solvent to improve immobilization of hydrophobic peptides.

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