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

Research output: Chapter in Book/Report/Conference proceedingChapter

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)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages405-416
Number of pages12
DOIs
StatePublished - 2021

Publication series

NameMethods in Molecular Biology
Volume2178
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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