Abstract
Co-immunoprecipitation (Co-IP) is one of the most broadly used methodologies to identify, validate, and characterize protein–protein interactions (PPIs) in native conditions. In this technique, a protein of interest is immunocaptured from a tissue lysate using a specific antibody. The immunocomplex containing the antibody, the antigen of interest, and the antigen-associated proteins is then precipitated by means of a protein A/G conjugated resin and analyzed by immunoblot. Interestingly, as PPIs may be extremely dynamic and/or labile, special attention to the antibodies and the lysis buffer used should be paid; therefore, the Co-IP method needs to be optimized for each specific PPI. Finally, an important question with this approach is the difficulty to differentiate physiological (real) interactors from nonspecific binders. Here, we describe the methodology to Co-IP two plasma membrane receptors from different brain areas (i.e., hippocampus and raphe nucleus) using the same set of antibodies.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Suzuki H (2006) Protein-protein interactions in the mammalian brain. J Physiol 575:373–377. https://doi.org/10.1113/jphysiol.2006.115717
Yao Z, Petschnigg J, Ketteler R, Stagljar I (2015) Application guide for omics approaches to cell signaling. Nat Chem Biol 11:387–397. https://doi.org/10.1038/nchembio.1809
Berggård T, Linse S, James P (2007) Methods for the detection and analysis of protein-protein interactions. Proteomics 7:2833–2842. https://doi.org/10.1002/pmic.200700131
Rao VS, Srinivas K, Sujini GN, Kumar GNS (2014) Protein-protein interaction detection: methods and analysis. Int J Proteom 2014:1–12. https://doi.org/10.1155/2014/147648
Markham K, Bai Y, Schmitt-Ulms G (2007) Co-immunoprecipitations revisited: an update on experimental concepts and their implementation for sensitive interactome investigations of endogenous proteins. Anal Bioanal Chem 389:461–473. https://doi.org/10.1007/s00216-007-1385-x
Borroto-Escuela DO, Romero-Fernandez W, Mudó G et al (2012) Fibroblast growth factor receptor 1-5-hydroxytryptamine 1A heteroreceptor complexes and their enhancement of hippocampal plasticity. Biol Psychiatry 71:84–91. https://doi.org/10.1016/j.biopsych.2011.09.012
Clark JD, Gebhart GF, Gonder JC et al (1997) Special report: the 1996 guide for the care and use of laboratory animals. ILAR J 38:41–48
Acknowledgments
This work was supported by Ministerio de Ciencia, Innovación y Universidades–Agencia Estatal de Investigación-FEDER-UE (SAF2017-87349-R MICIU/AEI/FEDER/UE) and Generalitat de Catalunya (2017SGR1604). We thank Centres de Recerca de Catalunya (CERCA) Programme/Generalitat de Catalunya for IDIBELL institutional support. We thank E. Castaño and B. Torrejón from the Scientific and Technical Services (SCT) group at the Bellvitge Campus of the University of Barcelona for their technical assistance.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Morató, X., Borroto-Escuela, D.O., Fuxe, K., Fernández-Dueñas, V., Ciruela, F. (2021). Co-Immunoprecipitation from Brain. In: Lujan, R., Ciruela, F. (eds) Receptor and Ion Channel Detection in the Brain. Neuromethods, vol 169. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1522-5_2
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1522-5_2
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1521-8
Online ISBN: 978-1-0716-1522-5
eBook Packages: Springer Protocols