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Optimization and implementation of entrapment: A novel immobilization technique for high performance affinity chromatography
The proper use of high performance affinity chromatography (HPAC) in the study of biologically-related systems requires that careful attention be paid to the nature in which the affinity ligand is incorporated into the stationary phase. Ideally, the behavior of the immobilized ligand should mimic the behavior of the ligand in its natural environment. Retaining the soluble form of the ligand, by avoiding covalent immobilization completely, is one effective way to retain the activity of the ligand. Previously, noncovalent immobilization techniques that do not modify the ligand of interest have included physical entrapment onto low performance supports. ^ This dissertation introduces an alternative method of entrapment onto supports that can withstand the high pressures and flow rates associated with HPAC. The entrapment method is based on the physical containment of a ligand in a polysaccharide-capped dihydrazide support. This method was optimized for maximum ligand content, and employed for the immobilization of several different ligands onto HPAC supports, all of which retained essentially 100% of their binding activity. These supports were used to study the binding affinities of a variety of drugs and the results were compared with previously established values obtained from studies using covalently immobilized ligands.^
Jackson, Abby Jo, "Optimization and implementation of entrapment: A novel immobilization technique for high performance affinity chromatography" (2011). ETD collection for University of Nebraska - Lincoln. AAI3449901.