Size Analysis of C9orf72 Dipeptide Repeat Proteins Expressed in Drosophila melanogaster Using Semidenaturing Detergent Agarose Gel Electrophoresis
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This chapter supplements Chapter 6 on sample preparation and analysis using an analytical ultracentrifuge with fluorescence detection. In this related chapter, we describe how semidenaturing detergent agarose gel electrophoresis can be used to complement the analytical ultracentrifugation work, often as a prelude to careful biophysical analysis to help screen conditions to improve the success of sedimentation velocity experiments. We describe preparation of crude lysates made using Drosophila melanogaster and provide a protocol giving detailed instructions for successful fractionation of protein aggregates using SDD-AGE. While limited in resolving power, this method can identify fractionation in three pools based on sample migration in the gel: that of a monomer or limiting small oligomer species; intermediate aggregation pools, which are typically heterogeneous, represented as high retention smears; and large-scale aggregation, found caught up in the wells.
Key wordsSemidenaturing detergent agarose gel electrophoresis Western blotting Protein aggregation Neurodegeneration Amyotrophic lateral sclerosis Frontotemporal dementia Drosophila melanogaster