Please use this identifier to cite or link to this item: https://doi.org/10.1016/j.ab.2012.01.020
Title: Immobilization of hydrophobic peptidic ligands to hydrophilic chromatographic matrix: A preconcentration approach
Authors: Gautam, S.
Loh, K.-C. 
Keywords: Hydrophilic matrix
Hydrophobic peptidic ligands
Immobilization
PEG crosslinker
Preconcentration
Issue Date: 15-Apr-2012
Source: Gautam, S.,Loh, K.-C. (2012-04-15). Immobilization of hydrophobic peptidic ligands to hydrophilic chromatographic matrix: A preconcentration approach. Analytical Biochemistry 423 (2) : 202-209. ScholarBank@NUS Repository. https://doi.org/10.1016/j.ab.2012.01.020
Abstract: This study presents a methodology for covalent attachment of hydrophobic peptidic ligands to hydrophilic chromatographic matrices with improved coupling efficiency. Preconcentration was introduced through the use of polyethylene glycol (PEG)-based crosslinkers. Immobilization of model hydrophobic peptide pep12 (ITLISSEGYVSS) to hydrophilic silica-amine matrix was investigated in the absence/presence of PEG-based linker. The effect of linker densities 14.2, 27.6, and 56.4 μmol/g beads on coupling efficiency was investigated. Whereas a ligand coupling efficiency of 67% was obtained in the absence of the linker, incorporating PEG-based linker at low densities allowed a 30% increase in the coupling efficiency. Although the heterobifunctional crosslinker, maleimide-PEG-NHS (N-hydroxysuccinimide) ester, can be used to couple thiol-bearing ligands to amine-functionalized matrices, no method is available for quenching free amine moieties on the matrix after ligand immobilization. The efficacy of acylating agents, acetyl chloride and oxalyl chloride, in blocking free amine groups when immobilizing the model peptide pep14 (CITLISSEGYVSSK) to silica-amine matrix using maleimide-PEG-NHS ester crosslinker was investigated. Because oxalyl chloride was nonreactive to maleimides, it allowed successful coupling of pep14 to the maleimide termini of the linkers. Adsorption studies between pep14-immobilized microspheres and human immunoglobulin M (hIgM) suggested retention of ligand activity and a 95% decrease in nonspecific binding of proteins to the matrix. © 2012 Elsevier Inc. All rights reserved.
Source Title: Analytical Biochemistry
URI: http://scholarbank.nus.edu.sg/handle/10635/64057
ISSN: 00032697
DOI: 10.1016/j.ab.2012.01.020
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