Size Analysis of C9orf72 Dipeptide Repeat Proteins Expressed in Drosophila melanogaster Using Semidenaturing Detergent Agarose Gel Electrophoresis

  • Nicole R. Cunningham
  • Bashkim Kokona
  • Jeanne M. Quinn
  • Robert FairmanEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2039)


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 words

Semidenaturing detergent agarose gel electrophoresis Western blotting Protein aggregation Neurodegeneration Amyotrophic lateral sclerosis Frontotemporal dementia Drosophila melanogaster 


  1. 1.
    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 72:245–256PubMedPubMedCentralGoogle Scholar
  2. 2.
    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 72:257–268PubMedPubMedCentralGoogle Scholar
  3. 3.
    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 525:129–133CrossRefGoogle Scholar
  4. 4.
    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 507:195–200CrossRefGoogle Scholar
  5. 5.
    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 278:49636–49643CrossRefGoogle Scholar
  6. 6.
    Halfmann R, Lindquist S (2008) Screening for amyloid aggregation by semi-denaturing detergent-agarose gel electrophoresis. J Vis Exp 17:838. Scholar
  7. 7.
    Holmberg M, Nollen EA (2013) Analyzing modifiers of protein aggregation in C. elegans by native agarose gel electrophoresis. Methods Mol Biol 1017:193–199CrossRefGoogle Scholar
  8. 8.
    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 56:4676–4688CrossRefGoogle Scholar
  9. 9.
    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 25:605–617CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Nicole R. Cunningham
    • 1
  • Bashkim Kokona
    • 1
  • Jeanne M. Quinn
    • 1
  • Robert Fairman
    • 1
    Email author
  1. 1.Department of BiologyHaverford CollegeHaverfordUSA

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