Abstract
A polypeptide chain can interact with other polypeptide chains and form stable and functional complexes called “oligomers.” Frequently, biochemical analysis of these complexes is made difficult by their great size. Traditionally, size exclusion chromatography, immunoaffinity chromatography, or immunoprecipitation techniques have been used to isolate oligomers. Components of these oligomers are then further separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and identified by immunoblotting with specific antibodies. Although they are sensitive, these techniques are not easy to perform and reproduce. The use of Tris-acetate polyacrylamide gradient gel electrophoresis allows the simultaneous analysis of proteins in the mass range of 10–500 kDa. We have used this characteristic together with cross-linking reagents to analyze the oligomerization of endogenous proteins with a single electrophoretic gel. We demonstrate how the oligomerization of p53, the pyruvate kinase isoform M2, or the heat shock protein 27 can be studied with this system. We also show how this system is useful for studying the oligomerization of large proteins such as clathrin heavy chain or the tuberous sclerosis complex. Oligomerization analysis is dependent on the cross-linker used and its concentration. All of these features make this system a very helpful tool for the analysis of protein oligomerization.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kim YE, Hipp MS, Bracher A, Hayer-Hart M, Hart FU (2013) Molecular chaperone functions in protein folding and proteostasis. Annu Rev Biochem 82:323–355
Matthews JM, Sunde M (2012) Dimers, oligomers, everywhere. Adv Exp Med Biol 747:1–18
Yang W, Lu Z (2013) Regulation and function of pyruvate kinase M2 in cancer. Cancer Lett 339:153–158
Iqbal MA, Gupta V, Gopinath P, Mazurek S, Bamezai RN (2014) Pyruvate kinase M2 and cancer: an updated assessment. FEBS Lett 588:2685–2692
Chène P (2001) The role of tetramerization in p53 function. Oncogene 20:2611–2617
Kawaguchi T, Kato S, Otsuka K, Watanabe G, Kumabe T, Tominaga T, Yoshimoto T, Ishioka C (2005) The relationship among p53 oligomer formation, structure and transcriptional activity using a comprehensive missense mutation library. Oncogene 24:6976–6981
Itahana Y, Ke H, Zhang Y (2009) p53 Oligomerization is essential for its C-terminal lysine acetylation. J Biol Chem 284:5158–5164
Ng HK, Chow BK (2015) Oligomerization of family B GPCRs: exploration in inter-family oligomer formation. Front Endocrinol (Lausanne) 6:10. doi:10.3389/fendo.2015.00010
Davison TS, Yin P, Nie E, Kay C, Arrowsmith CH (1998) Characterization of the oligomerization defects of two p53 mutants found in families with Li-Fraumeni and Li-Fraumeni-like syndrome. Oncogene 17:651–656
DiGiammarino EL, Lee AS, Cadwell C, Zhang W, Bothner B, Ribeiro RC, Zambetti G, Kriwacki RW (2002) A novel mechanism of tumorigenesis involving pH-dependent destabilization of a mutant p53 tetramer. Nat Struct Biol 9:12–16
Cubillos-Rojas M, Amair-Pinedo F, Tato I, Bartrons R, Ventura F, Rosa JL (2010) Simultaneous electrophoretic analysis of proteins of very high and low molecular mass using Tris-acetate polyacrylamide gels. Electrophoresis 31:1318–1321
Cubillos-Rojas M, Amair-Pinedo F, Tato I, Bartrons R, Ventura F, Rosa JL (2012) Tris-acetate polyacrylamide gradient gels for the simultaneous electrophoretic analysis of proteins of very high and low molecular mass. Methods Mol Biol 869:205–213
Krohn NM, Yanagisawa S, Grasser KD (2002) Specificity of the stimulatory interaction between chromosomal HMGB proteins and the transcription factor Dof2 and its negative regulation by protein kinase CK2-mediated phosphorylation. J Biol Chem 277:32438–32444
Acknowledgments
This study was supported by the Spanish Ministerio de Ciencia e Innovación Grant BFU2011-22498 and the Instituto de Salud Carlos III Grant RETIC, RD06/0020. T. Schneider was supported by a fellowship from the CAPES Foundation, Ministry of Education of Brazil. S. Sánchez-Tena was supported by a grant (PDJ 2013) from Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR), Generalitat de Catalunya, Spain. The authors would like to acknowledge networking support by the Proteostasis COST Action (BM1307).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Cubillos-Rojas, M., Schneider, T., Sánchez-Tena, S., Bartrons, R., Ventura, F., Rosa, J.L. (2016). Analysis of Protein Oligomerization by Electrophoresis. In: Matthiesen, R. (eds) Proteostasis. Methods in Molecular Biology, vol 1449. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3756-1_22
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3756-1_22
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3754-7
Online ISBN: 978-1-4939-3756-1
eBook Packages: Springer Protocols