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URL: https://dx.doi.org/DOI:10.17504/protocols.io.iiwccfe

Authors: Contreras-Treviño HI, Reyna-Rosas E, Padilla-Noriega L.

Summary: The procedures to determine the half-lives of proteins by pulse-chase analysis and cycloheximide blocking are well established. In the case of oligomeric proteins, these methods do not differentiate whether the stabilities of the monomeric versus oligomeric forms of the protein differ. This method describes how to determine the half-lives of oligomeric proteins that are stable in the presence of low concentrations of the detergent SDS by combining cycloheximide blocking with non-dissociating Western blot (Dutta et al., 2011). The addition of cycloheximide inhibits protein synthesis and non-dissociating Western blot uses low concentrations of SDS to separate stable oligomers from monomers. The amount of the protein of interest detected in samples taken at different times after adding cycloheximide can be used to determine the half-life of the oligomeric protein.ReferencesByk, L., Iglesias, N., De Maio, F., Gebhard, L., Rossi, M. and Gamarnik, A. (2016). Dengue virus genome uncoating requires ubiquitination. mBio, 7(3), pp.e00804-16.Dutta, D., Chattopadhyay, S., Bagchi, P., Halder, U., Nandi, S., Mukherjee, A., Kobayashi, N., Taniguchi, K. and Chawla-Sarkar, M. (2011). Active Participation of Cellular Chaperone Hsp90 in Regulating the Function of Rotavirus Nonstructural Protein 3 (NSP3). Journal of Biological Chemistry, 286(22), pp. 20065-20077.Elroy-Stein, O., Moss, B., 2001. Gene expression using the vaccinia virus/T7 RNA polymerase hybrid system. In: Current Protocols in Molecular Biology, chapter 16, unit16 9.Ward, G.A., Stover, C.K., Moss, B., Fuerst, T.R., 1995. Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells. Proceding of the Nationall Academy of Sciences U S A, 92, pp. 6773-6777.

Associated Publications: Contreras-Treviño HI, Reyna-Rosas E, León-Rodríguez R, Ruiz-Ordaz BH, Dinkova TD, Cevallos AM, Padilla-Noriega L (2017) Species A rotavirus NSP3 acquires its translation inhibitory function prior to stable dimer formation. PLoS ONE 12(7): e0181871. doi: 10.1371/journal.pone.0181871

Affiliations: Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico, Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico, Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico

External URL: https://doi.org/10.1371/journal.pone.0181871

Version: 1

Publication Date: 2017

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