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Quantitative Immunofluorescence to Measure Global Localized Translation

Quantitative Immunofluorescence to Measure Global Localized Translation

Published on: August 22, 2017

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

Charlotte Stadler¹, Marie Skogs, Hjalmar Brismar

¹School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), SE-106 91 Stockholm, Sweden.

Journal of Proteomics

|November 10, 2009

Summary

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Standardizing immunofluorescence microscopy fixation is crucial for proteome-wide studies. Paraformaldehyde cross-linking with Triton X-100 offers a reliable single protocol for consistent subcellular protein localization analysis.

Area of Science:

Cell Biology
Proteomics
Microscopy Techniques

Background:

Immunofluorescence microscopy is vital for analyzing protein expression and subcellular localization.
Standardized fixation protocols are needed for systematic, proteome-wide studies, as protein accessibility varies by compartment and composition.
Current methods require careful consideration of these factors, limiting broad applicability.

Purpose of the Study:

To identify a standardized fixation protocol for immunofluorescence microscopy applicable to a majority of human proteins.
To evaluate the effectiveness of different fixation methods across various subcellular localizations.
To establish a reliable protocol for proteome-wide protein localization studies.

Main Methods:

Tested six fixation protocols on 18 human proteins across 11 organelles in three cell lines.

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Protocols included alcohol dehydration (methanol, ethanol, isopropanol) and paraformaldehyde cross-linking.

Paraformaldehyde fixation was combined with detergent permeabilization using Triton X-100 or saponin.

Main Results:

Cross-linking fixation is essential for successful proteome-wide localization studies.
Paraformaldehyde cross-linking followed by Triton X-100 permeabilization proved effective for diverse proteins and localizations.
This combined method demonstrated suitability as a single, standardized protocol.

Conclusions:

Paraformaldehyde cross-linking with Triton X-100 permeabilization is a robust, standardized fixation protocol for immunofluorescence microscopy.
This method enables reliable, systematic proteome-wide analysis of protein localization.
The findings support the adoption of this protocol for broad applications in cell biology research.