Assessing Self-interaction of Mammalian Nuclear Proteins by Co-immunoprecipitation
Claudia Cattoglio,
Iryna Pustova,
Xavier Darzacq,
Robert Tjian,
Anders Hansen
Affiliations
Claudia Cattoglio
Department of Molecular and Cell Biology, University of California, Berkeley, CA, USALi Ka Shing Center for Biomedical and Health Sciences, Berkeley, CA, USA, CIRM Center of Excellence, University of California, Berkeley, CA, USA, Howard Hughes Medical Institute, University of California, Berkeley, CA, USA
Iryna Pustova
Department of Molecular and Cell Biology, University of California, Berkeley, CA, USALi Ka Shing Center for Biomedical and Health Sciences, Berkeley, CA, USA, CIRM Center of Excellence, University of California, Berkeley, CA, USA, Howard Hughes Medical Institute, University of California, Berkeley, CA, USA
Xavier Darzacq
Department of Molecular and Cell Biology, University of California, Berkeley, CA, USALi Ka Shing Center for Biomedical and Health Sciences, Berkeley, CA, USA, CIRM Center of Excellence, University of California, Berkeley, CA, USA
Robert Tjian
Department of Molecular and Cell Biology, University of California, Berkeley, CA, USALi Ka Shing Center for Biomedical and Health Sciences, Berkeley, CA, USA, CIRM Center of Excellence, University of California, Berkeley, CA, USA, Howard Hughes Medical Institute, University of California, Berkeley, CA, USA
Anders Hansen
Department of Molecular and Cell Biology, University of California, Berkeley, CA, USALi Ka Shing Center for Biomedical and Health Sciences, Berkeley, CA, USA, CIRM Center of Excellence, University of California, Berkeley, CA, USA, Howard Hughes Medical Institute, University of California, Berkeley, CA, USA
Protein-protein interactions constitute the molecular foundations of virtually all biological processes. Co-immunoprecipitation (CoIP) experiments are probably the most widely used method to probe both heterotypic and homotypic protein-protein interactions. Recent advances in super-resolution microscopy have revealed that several nuclear proteins such as transcription factors are spatially distributed into local high-concentration clusters in mammalian cells, suggesting that many nuclear proteins self-interact. These observations have further underscored the need for orthogonal biochemical approaches for testing if self-association occurs, and if so, what the mechanisms are. Here, we describe a CoIP protocol specifically optimized to test self-association of endogenously tagged nuclear proteins (self-CoIP), and to evaluate the role of nucleic acids in such self-interaction. This protocol has proven reliable and robust in our hands, and it can be used to test both homotypic and heterotypic (CoIP) protein-protein interactions.
