Isolation of Aggresomes and Other Large Aggregates

Anatoli B. Meriin1, Yan Wang1, Michael Y. Sherman1

1 Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.38
DOI:  10.1002/0471143030.cb0338s48
Online Posting Date:  September, 2010
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Upon permanent stresses and in various diseases, small protein aggregates may accumulate in cells and cause toxicity. A recently discovered protective system transports these aggregates to the centrosome location via microtubules, to form a large agglomerate of aggregates called the aggresome. Here, we describe a newly developed method for isolating aggresomes. This principle can also be used for purification of other large structures and even organelles. Curr. Protoc. Cell Biol. 48:3.38.1‐3.38.9. © 2010 by John Wiley & Sons, Inc.

Keywords: aggresome; aggregates; subcellular fractionation; yeast

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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Aggresomes from Yeast
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Isolation of Aggresomes from Yeast

  • Wild‐type yeast strain W303 (MATa ade2‐1 trp1‐1 leu2‐3,112 his3‐11,15 ura3‐52 can1‐100 ssd1‐d)
  • pYES2 vector (used as a negative control) or pYES2‐based plasmids for expression of 103QP or 103Q constructs under control of the Gal1 promoter (Meriin et al., ); 103QP encodes the full exon 1 of mutant (103 glutamines extension) htt tagged with FLAG at the N‐terminus and with EGFP at the C‐terminus; 103Q is the same construct missing the proline‐rich domain of the exon 1
  • Selective medium with 2% (w/v) glucose (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Selective medium with 2% (w/v) galactose (see recipe)
  • Lysis buffer (see recipe)
  • Glass beads (Sigma, cat. no. G8772)
  • Mouse anti‐FLAG monoclonal antibody (Sigma)
  • AffiniPure rabbit anti–mouse and goat anti–rabbit IgGs (H+L; Jackson ImmunoResearch Laboratories)
  • 50% (w/v) sucrose in lysis buffer
  • 30°C incubator
  • Bullet blender (NextAdvance)
  • 50‐ml Sephacryl S‐400 HR 2.5 (dia.) × 10–cm gel filtration column
  • Fluorescent microscope
  • 15‐ml test tubes
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Literature Cited

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