Aggregation Profiling of C9orf72 Dipeptide Repeat Proteins Transgenically Expressed in Drosophila melanogaster Using an Analytical Ultracentrifuge Equipped with Fluorescence Detection.

Abstract

The recent development of a fluorescence detection system for the analytical ultracentrifuge has allowed for the characterization of protein size and aggregation in complex mixtures. Protocols are described here to analyze protein aggregation seen in various human neurodegenerative diseases as they are presented in transgenic animal model systems. Proper preparation of crude extracts in appropriate sample buffers is critical for success in analyzing protein aggregation using sedimentation velocity methods. Furthermore, recent advances in sedimentation velocity analysis have led to data collection using single multispeed experiments, which may be analyzed using a wide distribution analysis approach. In this chapter, we describe the use of these new sedimentation velocity methods for faster determination of a wider range of sizes. In Chapter 7 of this book, we describe how agarose gel electrophoresis can be used to complement the analytical ultracentrifugation work, often as a prelude to careful biophysical analysis to help screen conditions in order to improve the success of sedimentation velocity experiments.