Equilibrium analysis of high affinity interactions using BIACORE

被引:92
作者
Myszka, DG [1 ]
Jonsen, MD [1 ]
Graves, BJ [1 ]
机构
[1] Univ Utah, Huntsman Canc Inst, Dept Oncol Sci, Salt Lake City, UT 84112 USA
关键词
biosensor; Ets-1; transcription factor; protein-DNA; interactions; surface plasmon resonance;
D O I
10.1006/abio.1998.2937
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
BIACORE biosensors are useful for measuring reaction kinetics and calculating affinity constants for macromolecular interactions. However, one drawback with the dow system used in these instruments is that the standard injection procedures limit the amount of time available to collect association-phase data. This is especially problematic during equilibrium analysis of high affinity interactions. Using protein-DNA interactions as a model system, we demonstrate a simple method for overcoming this limitation. By placing the analyte directly into the running buffer we were able to deliver a continuous supply of protein to the sensor surfaces for greater than 12 h at a time. Complete equilibrium binding profiles were generated by changing the concentration of analyte and allowing the surface reactions to reequilibrate. Analyte concentrations were also decreased to demonstrate that the binding reactions were fully reversible. This method of analysis is a simple and convenient way of directly measuring equilibrium dissociation constants for very high affinity interactions. (C) 1998 Academic Press.
引用
收藏
页码:326 / 330
页数:5
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