title: Analysis of biomolecular interactions using affinity microcolumns: a review.
abstract: Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored. The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites. It is further demonstrated how information on competition or displacement effects can be obtained by these methods.
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA.
authors.forename: Xiwei
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA.
authors.forename: Sandya
authors.initials: S
authors.lastname: Beeram
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Podariu
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Matsuda
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA.
authors.forename: Erika L
authors.initials: EL
authors.lastname: Pfaunmiller
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: White
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA.
authors.forename: NaTasha
authors.initials: N
authors.lastname: Carter
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA. Electronic address: dhage1@unl.edu.
authors.forename: David S
authors.initials: DS
authors.lastname: Hage
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry
chemical_list: D011506:Proteins
keywords: Affinity microcolumns; Biointeraction analysis; Frontal affinity chromatography; High-performance affinity chromatography; Ultrafast affinity extraction; Zonal elution