Monolithic columns with immobilized monomeric avidin: preparation and application for affinity chromatography |
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Authors: | Jens?Spro? Email author" target="_blank">Andrea?SinzEmail author |
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Institution: | (1) Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany; |
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Abstract: | A poly(glycidyl methacrylate-co-acrylamide-co-ethylene dimethacrylate) monolith and a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith were prepared in fused silica capillaries (100 μm ID) and modified with monomeric avidin
using the glutaraldehyde technique. The biotin binding capacity of monolithic affinity columns with immobilized monomeric
avidin (MACMAs) was determined by fluorescence spectroscopy using biotin (5-fluorescein) conjugate, as well as biotin- and
fluorescein-labeled bovine serum albumin (BSA). The affinity columns were able to bind 16.4 and 3.7 μmol biotin/mL, respectively.
Columns prepared using the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith retained 7.1 mg BSA/mL, almost six times more than commercially available monomeric avidin
beads. Protocols based on MALDI-TOF mass spectrometry monitoring were optimized for the enrichment of biotinylated proteins
and peptides. A comparison of enrichment efficiencies between MACMAs and commercially available monomeric avidin beads yielded
superior results for our novel monolithic affinity columns. However, the affinity medium presented in this work suffers from
a significant degree of nonspecific binding, which might hamper the analysis of more complex mixtures. Further modifications
of the monolith’s surface are envisaged for the future development of monoliths with improved enrichment characteristics. |
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