Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting
| Autor: | Hélène Obriot, Virginie Dutoit-Lefevre, Didier Lefranc, David Blum, Malika Hamdane, Luc Buée, Francisco-Jose Fernandez-Gomez, Maxime Derisbourg, Vincent Deramecourt, Valérie Mitchell, Sylvie Burnouf, Sabiha Eddarkaoui, Valérie Buée-Scherrer, Fanny Jumeau, Nicolas Sergeant, Helene Tran |
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| Rok vydání: | 2014 |
| Předmět: |
Proteome
Difference gel electrophoresis General Chemical Engineering Immunoblotting Nerve Tissue Proteins Computational biology Biology Proteomics General Biochemistry Genetics and Molecular Biology Two-Dimensional Difference Gel Electrophoresis Mice Protein purification Animals Humans Polyacrylamide gel electrophoresis Fluorescent Dyes Gel electrophoresis Brain Chemistry General Immunology and Microbiology General Neuroscience Brain Carbocyanines Molecular biology Blot Isoelectric point Neuroscience |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| Popis: | Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets. |
| Databáze: | OpenAIRE |
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