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

Annette C Moser; Benjamin White; Frank A Kovacs

doi:10.1007/978-1-0716-0775-6_26

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

Methods Mol Biol. 2021:2178:405-416. doi: 10.1007/978-1-0716-0775-6_26.

Authors

Annette C Moser¹, Benjamin White², Frank A Kovacs²

Affiliations

¹ Chemistry Department, University of Nebraska at Kearney, Kearney, NE, USA. moserac@unk.edu.
² Chemistry Department, University of Nebraska at Kearney, Kearney, NE, USA.

PMID: 33128763
DOI: 10.1007/978-1-0716-0775-6_26

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.

Keywords: Affinity chromatography; Binding constant; Enzyme immobilization; Frontal analysis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Chromatography, Affinity*
Histidine / chemistry*
Histidine / genetics
Nickel / chemistry*
Recombinant Fusion Proteins / chemistry*
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / isolation & purification*

Substances

Recombinant Fusion Proteins
polyhistidine
Histidine
Nickel