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Equilibrium analysis of high affinity interactions using BIACORE
D G Myszka1, M D Jonsen, B J Graves
1Huntsman Cancer Institute, University of Utah, 15 N. 2030 E., Room 2100, Salt Lake City, Utah, 84112-5330, USA. dmyszka@hci.utah.edu
Analytical Biochemistry
|January 12, 1999
Summary
This study presents a new method for BIACORE biosensors to measure high-affinity interactions. By adding analytes to the running buffer, researchers can collect equilibrium binding data for over 12 hours, improving kinetic analysis.
Area of Science:
- Biophysical Chemistry
- Biomolecular Interaction Analysis
Background:
- BIACORE biosensors measure reaction kinetics and affinity constants for macromolecular interactions.
- Standard injection methods limit association-phase data collection, especially for high-affinity interactions.
Purpose of the Study:
- To develop a method to overcome limitations in BIACORE biosensor association-phase data collection.
- To enable equilibrium analysis of high-affinity interactions.
Main Methods:
- Utilized protein-DNA interactions as a model system.
- Placed analyte directly into the running buffer for continuous supply to sensor surfaces (>12 hours).
- Generated complete equilibrium binding profiles by altering analyte concentration and allowing reequilibration.
Main Results:
- Demonstrated a simple and convenient method for BIACORE analysis.
- Successfully collected association-phase data for extended periods.
- Generated complete equilibrium binding profiles and confirmed reversibility of binding reactions.
Conclusions:
- The described method overcomes limitations of standard BIACORE injection procedures for high-affinity interactions.
- Enables direct measurement of equilibrium dissociation constants for very high affinity interactions.
- Provides a robust approach for detailed kinetic and affinity analysis of biomolecular interactions.