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Measuring Binding Constants of His-Tagged Proteins Using Affinity Chromatography and Ni-NTA-Immobilized Enzymes

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Protein Downstream Processing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1129))

Abstract

Affinity chromatography is one way to measure the binding constants of a protein–ligand interaction. Here we describe a method of measuring a binding constant using Ni-NTA resin to immobilize a His-tagged enzyme and the method of frontal analysis. While other methods of immobilization are possible, using the strong affinity interaction between His-tagged proteins and Ni-NTA supports results in a fast, easy, and gentle method of immobilization. Once the affinity support is created, frontal analysis can be used to measure the binding constant between the protein and various analytes.

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Acknowledgements

This work was supported by the UNK Summer Student Research Program, the UNK Undergraduate Research Fellows Program, and the UNK Chemistry Department.

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Authors and Affiliations

  1. Chemistry Department, University of Nebraska at Kearney, 905 West 25th Street, Kearney, NE, 68849-1150, USA

    Annette C. Moser

  2. Chemistry Department, University of Nebraska at Kearney, Kearney, NE, USA

    Benjamin White & Frank A. Kovacs

Authors
  1. Annette C. Moser
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  2. Benjamin White
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  3. Frank A. Kovacs
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Corresponding author

Correspondence to Annette C. Moser .

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Editors and Affiliations

  1. Laboratory of Enzyme Technology, Agricultural University of Athens Department of Agr. Biotechnology, Athens, Greece

    Nikolaos E. Labrou

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Moser, A.C., White, B., Kovacs, F.A. (2014). Measuring Binding Constants of His-Tagged Proteins Using Affinity Chromatography and Ni-NTA-Immobilized Enzymes. In: Labrou, N. (eds) Protein Downstream Processing. Methods in Molecular Biology, vol 1129. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-977-2_30

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  • DOI: https://doi.org/10.1007/978-1-62703-977-2_30

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-976-5

  • Online ISBN: 978-1-62703-977-2

  • eBook Packages: Springer Protocols

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