Analysis of proteins in solution using affinity capillary electrophoresis.
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.
Heegaard
Niels H H
NH
Department of Autoimmunology, Statens Serum Institut, Copenhagen S, Denmark.
Schou
Christian
C
Ostergaard
Jesper
J
D001665:Binding Sites; D002021:Buffers; D019075:Electrophoresis, Capillary / Q000379:methods*; D008024:Ligands; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012996:Solutions
D002021:Buffers; D008024:Ligands; D011506:Proteins; D012996:Solutions