title: Measuring Protein-Protein Interactions Using Biacore.
abstract: The use of optical biosensors for studying macromolecular interactions is gaining increasing popularity. In one study, 1514 papers that involved the application of biosensor data were identified for the year 2009 alone (Rich and Myszka, J Mol Recognit 24:892-914, 2011), the sheer volume and variety of which present a daunting task for the burgeoning biosensor user to accumulate and decipher. This chapter is designed to provide the reader with the tools necessary to prepare, design, and efficiently execute a kinetic experiment on Biacore. It is written to guide the Biacore user through basic theory, system maintenance, and assay setup while also offering some practical tips that we find useful for Biacore-based studies. Many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To highlight these procedures, this protocol describes the kinetic characterization of single chain Fv (scFv) antibody fragments from crude bacterial lysates using an antibody affinity capture approach. Even though we specifically describe the capture of HA-tagged scFv antibody fragments to an anti-HA tag monoclonal antibody-immobilized surface prior to kinetic analysis, the same methodologies are universally applicable and can be used for practically any affinity pair and most Biacore systems.
authors.affiliation: Biomedical Diagnostics Institute, Dublin City University, Dublin 9, Ireland.|School of Biotechnology, Dublin City University, Dublin 9, Ireland.|National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland.
authors.forename: Paul
authors.initials: P
authors.lastname: Leonard
authors.affiliation: Biomedical Diagnostics Institute, Dublin City University, Dublin 9, Ireland.|School of Biotechnology, Dublin City University, Dublin 9, Ireland.|National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland.
authors.forename: Stephen
authors.initials: S
authors.lastname: Hearty
authors.affiliation: Biomedical Diagnostics Institute, Dublin City University, Dublin 9, Ireland.
authors.forename: Hui
authors.initials: H
authors.lastname: Ma
authors.affiliation: Biomedical Diagnostics Institute, Dublin City University, Dublin 9, Ireland. Richard.okennedy@dcu.ie.|School of Biotechnology, Dublin City University, Dublin 9, Ireland. Richard.okennedy@dcu.ie.|National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland. Richard.okennedy@dcu.ie.
authors.forename: Richard
authors.initials: R
authors.lastname: O'Kennedy
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods
chemical_list: D002352:Carrier Proteins; D008024:Ligands; D011506:Proteins
keywords: Antibody; Biacore; Biosensor analysis; Kinetics; Protein–protein interactions; Surface plasmon resonance