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Immobilized Metal Affinity Chromatography of Phosphorylated Proteins Using High Performance Sorbents

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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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Acknowledgments

The authors acknowledge the financial support of the Ministry of Education of the Czech Republic (grant No. MSM 0021620806 and project No. LC 06044) and thank Prof. Marie Ticha from the Department of Biochemistry and the Institute of Pathophysiology and the Center of Experimental Hematology at Charles University in Prague for careful revision of the manuscript.

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Authors and Affiliations

  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

    Lenka Novotna & Zdenka Kucerova

  2. Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 16206, Prague 6, Czech Republic

    Martin Hruby & Milan J. Benes

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  1. Lenka Novotna
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  2. Martin Hruby
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  3. Milan J. Benes
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  4. Zdenka Kucerova
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Correspondence to Lenka Novotna.

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Novotna, L., Hruby, M., Benes, M.J. et al. Immobilized Metal Affinity Chromatography of Phosphorylated Proteins Using High Performance Sorbents. Chroma 68, 381–386 (2008). https://doi.org/10.1365/s10337-008-0727-y

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  • DOI: https://doi.org/10.1365/s10337-008-0727-y

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