Optical Biosensing: Kinetics of Protein A-IGG Binding Using Biolayer Interferometry
Department
Chemistry and Biochemistry
Document Type
Article
Publication Date
12-1-2010
Abstract
An undergraduate biochemistry laboratory experiment has been developed using biolayer interferometry (BLI), an optical biosensing technique similar to surface plasmon resonance (SPR), in which students obtain and analyze kinetic data for a protein-protein interaction. Optical biosensing is a technique of choice to determine kinetic and affinity constants for biomolecular interactions. Measurements can be made in real-time without labels, making biosensing particularly appropriate for the teaching laboratory. In the described exercise, students investigate the kinetics of Protein A-human Immunoglobin G binding under conditions that mimic simple 1:1 binding. Students prepare appropriate serial dilutions of IgG and set up a microplate for the experiment by aliquotting biotinylated Protein A, buffer, and IgG solutions. A commercial BLI sensor, the FortéBio Octet QK, is used to measure binding. While data are collected students prepare a spreadsheet with which they will simulate the data to determine kon, koff, and KD. Raw data from the sensor are then exported to the spreadsheets for analysis. Optimized experiment timing, regeneration methods and other parameters are described to increase throughput and reduce cost. The experiment is readily adaptable to other biosensing platforms such as SPR instruments.
Journal Title
Biochemistry and Molecular Biology Education
Journal ISSN
1539-3429
Volume
38
Issue
6
First Page
400
Last Page
407
Digital Object Identifier (DOI)
10.1002/bmb.20442