Measuring binding constants of his-tagged proteins using affinity chromatography and Ni-NTA-immobilized enzymes

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)423-434
Number of pages12
JournalMethods in Molecular Biology
Volume1129
DOIs
StatePublished - Jan 1 2014

Fingerprint

Immobilized Enzymes
Affinity Chromatography
Immobilization
Proteins
Gi-Go GTP-Binding Protein alpha Subunits
Enzymes

Keywords

  • Affinity chromatography
  • Binding constant
  • Enzyme immobilization
  • Frontal analysis

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

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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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AB - 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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