Abstract
Proteins often do not function as single substances but rather as team players in a dynamic network. Growing evidence shows that protein–protein interactions are crucial in many biological processes in living cells. Genetic (such as yeast two-hybrid, Y2H) and biochemical (such as co-immunoprecipitation, co-IP) methods are the methods commonly used at the beginning of a study to identify the interacting proteins. Immunoprecipitation (IP), a method using a target protein-specific antibody in conjunction with Protein A/G affinity beads, is a powerful tool to identify molecules that interact with specific proteins. Therefore, co-IP is considered to be one of the standard methods of identifying or confirming the occurrence of protein–protein interaction events in vivo. Co-IP experiments can identify proteins via direct or indirect interactions or in a protein complex. Here, we use Agrobacterium type VI secretion system (T6SS) sheath components TssB-TssC41 interaction as an example to describe the principle, procedure, and experimental problems of co-IP.
References
Kessler SW (1975) Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol 115:1617–1624
Kessler SW (1976) Cell membrane antigen isolation with the staphylococcal protein A-antibody adsorbent. J Immunol 117:1482–1490
ThermoFisher (2009) Immunoprecipitation (IP) technical guide and protocols. https://tools.thermofisher.com/content/sfs/brochures/TR0064-Immunoprecipitation-guide.pdf.
Lee C (2007) Coimmunoprecipitation assay. Methods Mol Biol 362:401–406
Aebersold R, Mann M (2003) Mass spectrometry-based proteomics. Nature 422:198–207
Gevaert K, Vandekerckhove J (2000) Protein identification methods in proteomics. Electrophoresis 21:1145–1154
Atmakuri K, Cascales E, Christie PJ (2004) Energetic components VirD4, VirB11 and VirB4 mediate early DNA transfer reactions required for bacterial type IV secretion. Mol Microbiol 54:1199–1211
Atmakuri K, Cascales E, Burton OT, Banta LM, Christie PJ (2007) Agrobacterium ParA/MinD-like VirC1 spatially coordinates early conjugative DNA transfer reactions. EMBO J 26:2540–2551
Anderson LB, Hertzel AV, Das A (1996) Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex. Proc Natl Acad Sci U S A 93:8889–8894
Lin JS, Ma LS, Lai EM (2013) Systematic dissection of the Agrobacterium type VI secretion system reveals machinery and secreted components for subcomplex formation. PLoS One 8:e67647
Ma LS, Narberhaus F, Lai EM (2012) IcmF family protein TssM exhibits ATPase activity and energizes type VI secretion. J Biol Chem 287:15610–15621
Elion EA (2007) Detection of protein-protein interactions by coprecipitation. Curr Protoc Immunol Chapter 8:Unit 8.7
Cascales E, Christie PJ (2004) Definition of a bacterial type IV secretion pathway for a DNA substrate. Science 304:1170–1173
Acknowledgements
This work was supported by a research grant from the Taiwan Ministry of Science and Technology (MOST 104-2311-B-001-025 -MY3) to E.M. Lai. J.S. Lin is the recipient of postdoctoral fellowships from Academia Sinica.
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Lin, JS., Lai, EM. (2017). Protein–Protein Interactions: Co-Immunoprecipitation. In: Journet, L., Cascales, E. (eds) Bacterial Protein Secretion Systems. Methods in Molecular Biology, vol 1615. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7033-9_17
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DOI: https://doi.org/10.1007/978-1-4939-7033-9_17
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