Analysis of Protein Oligomeric Species by Sucrose Gradients.
Protein misfolding, aggregation, and accumulation are a common hallmark in various neurodegenerative diseases. Invariably, the process of protein aggregation is associated with both a loss of the normal biological function of the protein and a gain of toxic function that ultimately leads to cell death. The precise origin of protein cytotoxicity is presently unclear but the predominant theory posits that smaller oligomeric species are more toxic than larger aggregated forms. While there is still no consensus on this subject, this is a central question that needs to be addressed in order to enable the design of novel and more effective therapeutic strategies. Accordingly, the development and utilization of approaches that allow the biochemical characterization of the formed oligomeric species in a given cellular or animal model will enable the correlation with cytotoxicity and other parameters of interest.Here, we provide a detailed description of a low-cost protocol for the analysis of protein oligomeric species from both yeast and mammalian cell lines models, based on their separation according to sedimentation velocity using high-speed centrifugation in sucrose gradients. This approach is an adaptation of existing protocols that enabled us to overcome existing technical issues and obtain reliable results that are instrumental for the characterization of the types of protein aggregates formed by different proteins of interest in the context of neurodegenerative disorders.
Instituto de Medicina Molecular, Lisboa, Portugal. stenreiro@fmns.unl.pt.|Faculdade de Ciências Médicas, CEDOC-Chronic Diseases Research Center, NOVA Medical School, Universidade Nova de Lisboa, Rua Câmara Pestana n° 6, Edifício CEDOC II, Lisboa, 1150-082, Portugal. stenreiro@fmns.unl.pt.
Sandra
S
Tenreiro
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, Oeiras, 2780-157, Portugal.
Diana
D
Macedo
Instituto de Medicina Molecular, Lisboa, Portugal.
Zrinka
Z
Marijanovic
Faculdade de Ciências Médicas, CEDOC-Chronic Diseases Research Center, NOVA Medical School, Universidade Nova de Lisboa, Rua Câmara Pestana n° 6, Edifício CEDOC II, Lisboa, 1150-082, Portugal. touteir@gwdg.de.|Instituto de Fisiologia, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal. touteir@gwdg.de.|Department of NeuroDegeneration and Restorative Research, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Göttingen, Göttingen, Germany. touteir@gwdg.de.
Tiago Fleming
TF
Outeiro
D000818:Animals; D002460:Cell Line; D002499:Centrifugation, Density Gradient / Q000379:methods*; D006801:Humans; D019636:Neurodegenerative Diseases / Q000378:metabolism; D066329:Protein Aggregates; D017510:Protein Folding; D055503:Protein Multimerization; D013395:Sucrose / Q000737:chemistry*
D066329:Protein Aggregates; D013395:Sucrose
High-speed centrifugation; Mammalian cell lines; Neurodegenerative diseases; Oligomers; Protein aggregates; Protein misfolding; Proteinopathies; Sucrose gradients; Velocity sedimentation; Yeast model