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Updated: Sep 29, 2026

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study
Published on: August 16, 2019
Cyanogen bromide activation and coupling of ligands to diol-containing silica for high-performance affinity
Luis A Jurado1, James Mosley, Harry W Jarrett
1Department of Biochemistry, University of Tennessee, Memphis 38163, USA.
Abstract:
To obtain silica supports for high-performance affinity chromatography, a method of preparing CNBr-activated diol-silica under anhydrous conditions was developed. Activation of the silane-derived hydroxyls with cyanogen bromide and triethylamine was optimized and demonstrated to efficiently couple several amino ligands (tryptophan, 6-aminohexyl-Cibacron Blue, and DNA). Sonication and vacuum degassing, a procedure used to remove air from the silica beads, increased activation. Coupling of an amino ligand under slightly basic conditions (pH 8.0) gave the highest yield. The linkage between the immobilized ligands and silica was stable from pH 2-10. Anhydrous acetone was the most effective solvent for activation but dimethylformamide and 2-propanol were also good choices. The high-performance affinity chromatography columns obtained by coupling sequence-specific DNA binding sequences for Lac repressor-beta-galactosidase fusion protein were compared to affinity columns obtained by coupling the same DNA element to Sepharose beads; 5 microm silica gave the best performance.
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