A single fixation protocol for proteome-wide immunofluorescence localization studies.

Immunofluorescence microscopy is a valuable tool for analyzing protein expression and localization at a subcellular level thus providing information regarding protein function, interaction partners and its role in cellular processes. When performing sample fixation, parameters such as difference in accessibility of proteins present in various cellular compartments as well as the chemical composition of the protein to be studied, needs to be taken into account. However, in systematic and proteome-wide efforts, a need exists for standard fixation protocol(s) that works well for the majority of all proteins independent of subcellular localization. Here, we report on a study with the goal to find a standardized protocol based on the analysis of 18 human proteins localized in 11 different organelles and subcellular structures. Six fixation protocols were tested based on either dehydration by alcohols (methanol, ethanol or iso-propanol) or cross-linking by paraformaldehyde followed by detergent permeabilization (Triton X-100 or saponin) in three human cell lines. Our results show that cross-linking is essential for proteome-wide localization studies and that cross-linking using paraformaldehyde followed by Triton X-100 permeabilization successfully can be used as a single fixation protocol for systematic studies.
AuthorsCharlotte Stadler, Marie Skogs, Hjalmar Brismar, Mathias Uhlén, Emma Lundberg
JournalJournal of proteomics (J Proteomics) Vol. 73 Issue 6 Pg. 1067-78 (Apr 18 2010) ISSN: 1876-7737 [Electronic] Netherlands
PMID19896565 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright 2009 Elsevier B.V. All rights reserved.
Chemical References
  • Detergents
  • Polymers
  • Proteome
  • Ribosome Inactivating Proteins, Type 1
  • Formaldehyde
  • Octoxynol
  • saporin
  • Peptide Hydrolases
  • paraform
  • Cell Line, Tumor
  • Cytoskeleton (metabolism)
  • Detergents (pharmacology)
  • Endoplasmic Reticulum (metabolism)
  • Formaldehyde (chemistry)
  • Golgi Apparatus (metabolism)
  • Humans
  • Microscopy, Fluorescence (methods)
  • Mitochondria (metabolism)
  • Octoxynol (pharmacology)
  • Peptide Hydrolases (chemistry)
  • Polymers (chemistry)
  • Proteome
  • Proteomics (methods)
  • Ribosome Inactivating Proteins, Type 1 (pharmacology)

Join CureHunter, for free Research Interface BASIC access!

Take advantage of free CureHunter research engine access to explore the best drug and treatment options for any disease. Find out why thousands of doctors, pharma researchers and patient activists around the world use CureHunter every day.
Realize the full power of the drug-disease research network!

Choose Username:
Verify Password: