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Analysis of Proteins in Solution Using Affinity
Capillary Electrophoresis
Niels H. H. Heegaard, Christian Schou, and Jesper Østergaard
Summary
Analysis of protein interactions by means of capillary electrophoresis (CE) has
unique challenges and rewards. The choice of analysis conditions, especially involving
electrophoresis buffers, are crucial and not universal for protein analysis. If conditions for
analysis can be worked out, it is possible to utilize CE quantitatively and qualitatively to
characterize protein-ligand binding involving unmodified molecules in solution and taking
place under physiological conditions. This chapter deals with the most important practical
considerations in capillary electrophoretic affinity approaches, affinity CE (ACE). The text
emphasizes the most critical factors for successful analyses and has application examples
illustrating various types of information offered by ACE-based studies. Also included are
step-by-step accounts of the two main classes of experimental design: the pre-equilibration
ACE (in the form of CE-frontal analysis (CE-FA)) and mobility shift ACE together with
examples of their use. The ACE approaches for binding assays of proteins should be
considered when the biological material is scarce, when any kind of labeling is not possible
or desired, when the interacting molecules are the same size and when rapid and simple
method development is a priority.
Key Words: Affinity capillary electrophoresis; binding assay; analytical conditions;
pre-equilibration ACE; mobility shift ACE.
From: Methods in Molecular Biology, vol. 421: Affinity Chromatography: Methods and Protocols, Second Edition
Edited by: M. Zachariou © Humana Press, Totowa, NJ
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