title: Measuring binding constants of His-tagged proteins using affinity chromatography and Ni-NTA-immobilized enzymes.
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.
authors.affiliation: Chemistry Department, University of Nebraska at Kearney, 905 West 25th Street, Kearney, NE, 68849-1150, USA, moserac@unk.edu.
authors.forename: Annette C
authors.initials: AC
authors.lastname: Moser
authors.forename: Benjamin
authors.initials: B
authors.lastname: White
authors.forename: Frank A
authors.initials: FA
authors.lastname: Kovacs
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry*; D009532:Nickel / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D011506:Proteins; D006639:Histidine; D009532:Nickel