Electrophoretic methods for studying protein-protein interactions.
Protein-protein interactions are involved in many biological processes ranging from DNA replication, to signal transduction, to metabolism control, to viral assembly. The understanding of those interactions would allow the effective design of new drugs and further manipulation of those interactions. Several useful analytical methods are available for the study of protein-protein binding, and among them, electrophoresis is commonly used. We describe two types of electrophoresis: gel electrophoresis and capillary electrophoresis. Gel electrophoresis is a well-established method used to study protein-protein interactions and includes overlay gel electrophoresis, charge shift method, band shift assay, countermigration electrophoresis, affinophoresis, affinity electrophoresis, rocket immunoelectrophoresis, and crossed immunoelectrophoresis. These techniques are briefly described along with their advantages and limitations. Capillary electrophoresis, on the other hand, is a relatively new method and affinity capillary electrophoresis has demonstrated its value in the measurement of binding constants, the estimation of kinetic rate constants, and the determination of stoichiometry of biomolecular interactions. It offers short analysis time, requires minute amounts of protein samples, usually involves no radiolabeled compounds, and, most importantly, is carried out in solution. We summarize the principles of affinity capillary electrophoresis for studying protein-protein interactions along with current limitations and describe in depth its application to the determination of stoichiometries of tight and weak binding protein-protein interactions. The protocol presented in the experimental section details the use of affinity capillary electrophoresis for the determination of stoichiometry of protein complexes.
Vergnon
A L
AL
Department of Chemistry, National Chung-Cheng University, Chia-Yi, Taiwan, 621.
Chu
Y H
YH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000936:Antigen-Antibody Complex; D004261:DNA Replication; D015195:Drug Design; D004586:Electrophoresis / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional / Q000379:methods; D051379:Mice; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012709:Serum Albumin / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D011506:Proteins; D012709:Serum Albumin