title: Size analysis of polyglutamine protein aggregates using fluorescence detection in an analytical ultracentrifuge.
abstract: Defining the aggregation process of proteins formed by poly-amino acid repeats in cells remains a challenging task due to a lack of robust techniques for their isolation and quantitation. Sedimentation velocity methodology using fluorescence detected analytical ultracentrifugation is one approach that can offer significant insight into aggregation formation and kinetics. While this technique has traditionally been used with purified proteins, it is now possible for substantial information to be collected with studies using cell lysates expressing a GFP-tagged protein of interest. In this chapter, we describe protocols for sample preparation and setting up the fluorescence detection system in an analytical ultracentrifuge to perform sedimentation velocity experiments on cell lysates containing aggregates formed by poly-amino acid repeat proteins.
authors.affiliation: Department of Biochemistry and Molecular Biology, Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia.
authors.forename: Saskia
authors.initials: S
authors.lastname: Polling
authors.forename: Danny M
authors.initials: DM
authors.lastname: Hatters
authors.forename: Yee-Foong
authors.initials: YF
authors.lastname: Mok
mesh_terms: D000818:Animals; D002460:Cell Line; D002499:Centrifugation, Density Gradient / Q000379:methods; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C097188:polyglutamine