Immobilized Metal Affinity Chromatography of Phosphorylated Proteins Using High Performance Sorbents |
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| Authors: | Lenka Novotna Martin Hruby Milan J Benes Zdenka Kucerova |
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| Institution: | 1. Institute of Pathophysiology and Center of Experimental Hematology, 1st Faculty of Medicine, Charles University in Prague, U Nemocnice 5, 12853, Prague 2, Czech Republic
2. Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 16206, Prague 6, Czech Republic
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| Abstract: | The interactions of two model phosphoproteins (porcine pepsin and ovalbumin) with two different immobilized metal affinity chromatography (IMAC) sorbents containing immobilized Fe3+, Ga3+, and UO2 2+ ions have been investigated under various conditions. Both proteins were adsorbed on immobilized uranyl ions under acidic conditions similar to those on immobilized Fe3+ and Ga3+ ions. The retained proteins could be released either by the presence of phosphate ions in the elution buffer (immobilized Ga3+ and Fe3+ ions) or by an increased pH (all tested immobilized ions). The IMAC sorbents employed could be used under the conditions of high-performance chromatography and are suitable for the separation and analysis of intact phosphoproteins. |
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