Abstract
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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
DeJesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M, Rutherford NJ et al (2011) Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72:245–256
Renton AE, Majounie E, Waite A, Simon-Sanchez J, Rollinson S, Gibbs JR et al (2011) A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72:257–268
Freibaum BD, Lu Y, Lopez-Gonzalez R, Kim NC, Almeida S, Lee KH et al (2015) GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature 525:129–133
Haeusler AR, Donnelly CJ, Periz G, Simko EA, Shaw PG, Kim MS et al (2014) C9orf72 nucleotide repeat structures initiate molecular cascades of disease. Nature 507:195–200
Kryndushkin DS, Alexandrov IM, Ter-Avanesyan MD, Kushnirov VV (2003) Yeast (PSI+) prion aggregates are formed by small Sup35 polymers fragmented by Hsp104. J Biol Chem 278:49636–49643
Halfmann R, Lindquist S (2008) Screening for amyloid aggregation by semi-denaturing detergent-agarose gel electrophoresis. J Vis Exp 17:838. https://doi.org/10.3791/838
Holmberg M, Nollen EA (2013) Analyzing modifiers of protein aggregation in C. elegans by native agarose gel electrophoresis. Methods Mol Biol 1017:193–199
Kim SA, D’Acunto VF, Kokona B, Hofmann J, Cunningham NR, Bistline EM et al (2017) Sedimentation velocity analysis with fluorescence detection of mutant huntingtin exon 1 aggregation in Drosophila melanogaster and Caenorhabditis elegans. Biochemistry 56:4676–4688
Kokona B, May CA, Cunningham NR, Richmond L, Garcia FJ, Durante JC et al (2016) Studying polyglutamine aggregation in Caenorhabditis elegans using an analytical ultracentrifuge equipped with fluorescence detection. Protein Sci 25:605–617
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Cunningham, N.R., Kokona, B., Quinn, J.M., Fairman, R. (2019). Size Analysis of C9orf72 Dipeptide Repeat Proteins Expressed in Drosophila melanogaster Using Semidenaturing Detergent Agarose Gel Electrophoresis. In: McManus, J. (eds) Protein Self-Assembly. Methods in Molecular Biology, vol 2039. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9678-0_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9678-0_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9677-3
Online ISBN: 978-1-4939-9678-0
eBook Packages: Springer Protocols