Published: Vol 3, Iss 3, Feb 5, 2013 DOI: 10.21769/BioProtoc.319 Views: 33838
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Abstract
Co-Immunoprecipitation (Co-IP) is the method used to pull down protein partners of a protein of interest using an antibody that specifically binds to this specific protein in order to test protein-protein interaction. “Pulled down” proteins can be analyzed by western blot for suspected protein partner, or by Mass spectrometry for high throughput protein partner identification. The advantage of this technique is that endogenous protein partners can be identified from cell lines that naturally express these factors.
This protocol is optimized for hard-to-extract nuclear proteins, e.g., that stick to the nuclei inclusion bodies / nucleosome complexes such as TLX1 and TLX3 (Dadi et al., 2012). Most often, these factors are not soluble when using classical protein extraction methods. We used to add nucleases in order to increase solubilization of protein complexes trapped within inclusion bodies; though the efficacy varies depending on the given protein and therefore has to be empirically determined.
Materials and Reagents
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Acknowledgments
Work in the PF laboratory is supported by institutional grants from 'Institut National de la Santé et de la Recherche Médicale' (Inserm) and 'Centre National de la Recherche Scientifique' (CNRS), and by dedicated grants from the Commission of the European Communities, the 'Agence Nationale de la Recherche' (ANR), the 'Institut National du Cancer' (INCa), the 'ITMO Cancer Alliance Nationale pour les Sciences de la Vie et de la Santé' (AVIESAN) and the 'Fondation Princesse Grace de la Principauté de Monaco'. S.D. was supported by fellowships from the ‘Ministère de l’Enseignement Supérieur et de la Recherche’, the ‘Fondation pour la Recherche Médicale’ (FRM), and the ‘Société Française d’Hématologie’ (SFH).
References
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Dadi, S., Payet-Bornet, D. and Ferrier, P. (2013). ImmunoPrecipitation of Nuclear Protein with Antibody Affinity Columns. Bio-protocol 3(3): e319. DOI: 10.21769/BioProtoc.319.
Category
Biochemistry > Protein > Immunodetection
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