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.
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.
Are these resins available as kits?
CarboxyLink Coupling Resin is available as a kit (#44899), which contains sufficient components to prepare five reusable affinity columns. The resin is also available separately (#20266, 25 ml). DADPA UltraLink is available only as a kit (#53154). The kits do not contain buffers for affinity purification.
How do I determine the degree of antibody coupling to the resin?
Protein samples can be quantified using Thermo Scientific Pierce BCA Protein Assay (#23225) or Coomassie Plus (Bradford) Protein Assay (#23236). See Tech Tip #9: Quantitate protein immobilized on a solid support. 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.
My peptide is not water-soluble; can I use other solvents for conjugation?
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 many purifications can be performed using the same affinity column?
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.
What is the difference between the Thermo Scientific CarboLink and CarboxyLink Resins?
The CarboxyLink Resin is for immobilizing carboxyl-containing biomolecules after EDC activation. CarboLink Resin is hydrazide-activated and immobilizes glycoproteins. It can also be used to immobilize steroids or other molecules containing ketones. Both resins are capable of coupling ligands via carboxylic acids using EDC (#22980). Both immobilization chemistries are available on UltraLink Resin as UltraLink Hydrazide (glycoproteins) and UltraLink DADPA (carboxyl) Resins.