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Immunofluorescence and fluorescent protein-tagging are complementary techniques with high correlation for subcellular investigation of the human proteome in mammalian cells
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-8993-048X
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(English)Article in journal (Other academic) Submitted
Abstract [en]

Imaging techniques such as immunofluorescence (IF) and expression of fluorescent protein (FP) fusions are widely used to investigate the subcellular distribution of proteins. Due to potential steric hindrance with FPs and fixation artifacts obtained during IF, the reliability of these two fundamental methods is often discussed. Here we report a systematic study of over 500 human proteins where the localizations obtained in live versus fixed cells using FPs and IF respectively have been compared. The results indicate that 80% of the analyzed proteins yield the same subcellular distribution, which is as high as that seen for FP tagging at either the N- and C-terminal. The localizations of 250 proteins, with no previous experimental data, were determined by the overlap of the two methods as applied here. The fraction of proteins located to multiple organelles is approximately 60% for both methods, indicating a complex subcellular protein organization. The result shows that IF and FP tagging are reliable techniques, both needed for a complete investigation of the subcellular human proteome.

National Category
Industrial Biotechnology
URN: urn:nbn:se:kth:diva-103628OAI: diva2:561089

QS 2012

Available from: 2012-10-17 Created: 2012-10-17 Last updated: 2012-10-17Bibliographically approved
In thesis
1. Towards subcellular localization of the human proteome using bioimaging
Open this publication in new window or tab >>Towards subcellular localization of the human proteome using bioimaging
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Since the publication of the complete sequence of the human genome in 2003 there has been great interest in exploring the functions of the proteins encoded by the genes. To reveal the function of each and every protein, investigation of protein localization at the subcellular level has become a central focus in this research area, since the localization and function of a protein is closely related. The objective of the studies presented in this doctoral thesis was to systematically explore the human proteome at the subcellular level using bioimaging and to develop techniques for validation of the results obtained.

A common imaging technique for protein detection is immunofluorescence (IF), where antibodies are used to target proteins in fixated cells. A fixation protocol suitable for large-scale IF studies was developed and optimized to work for a broad set of proteins. As the technique relies on antibodies, validation of their specificity to the target protein is crucial. A platform based on siRNA gene silencing in combination with IF was set-up to evaluate antibody specificity by quantitative image analysis before and after suppression of its target protein. As a proof of concept, the platform was then used for validation of 75 antibodies, proving it to be applicable for validation of antibodies in a systematic manner.

Because of the fixation, there is a common concern about how well IF data reflects the in vivo subcellular distribution of proteins. To address this, 500 proteins were tagged with green fluorescent protein (GFP) and used to compare protein localization results between IF to those achieved using GFP tagged proteins in live cells. It was concluded that protein localization data from fixated cells satisfactory represented the situation in vivo and together exhibit a powerful approach for confirming localizations of yet uncharacterized proteins.

Finally, a global analysis based on IF data of approximately 20 % of the human proteome was performed, providing a first overview of the subcellular landscape in three different cell lines. It was found that the intracellular distribution of proteins is complex, with many proteins occurring in several organelles. The results also confirmed the close relationship between protein function and localization, which in a way further strengthens the accuracy of the IF approach for detection of proteins at the subcellular level.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. 60 p.
Trita-BIO-Report, ISSN 1654-2312 ; 12:19
Antibody, antibody validation, automated image analysis, automated microscopy, cell line, confocal microscopy, fixation, green fluorescent protein (GFP), immunofluorescence (IF), organelle, protein expression, siRNA, subcellular localization
National Category
Biochemistry and Molecular Biology
urn:nbn:se:kth:diva-103616 (URN)978-91-7501-483-8 (ISBN)
Public defence
2012-11-09, MTC, Karolinska institutet, Stockholm, 10:00 (English)
The Human Protein Atlas
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience

QC 20121017

Available from: 2012-10-17 Created: 2012-10-16 Last updated: 2013-04-15Bibliographically approved

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Stadler, CharlotteUhlén, MathiasLundberg, Emma
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Proteomics (closed 20130101)Science for Life Laboratory, SciLifeLab
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