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A single fixation protocol for proteome-wide immunofluorescence localization studies
KTH, School of Biotechnology (BIO), Proteomics.
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0002-2998-3077
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.ORCID iD: 0000-0003-0578-4003
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0001-8993-048X
Show others and affiliations
2010 (English)In: Journal of Proteomics, ISSN 1874-3919, Vol. 73, no 6, 1067-1078 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2010. Vol. 73, no 6, 1067-1078 p.
Keyword [en]
Antibody, Confocal microscopy, Fixation, Immunofluorescence, Organelle, Permeabilization
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-29841DOI: 10.1016/j.jprot.2009.10.012ISI: 000277763800004Scopus ID: 2-s2.0-77950516661OAI: oai:DiVA.org:kth-29841DiVA: diva2:399858
Note
QC 20110223 3rd EuPA Congress, Stockholm, SWEDEN, JUN 14-17, 2009Available from: 2011-02-23 Created: 2011-02-17 Last updated: 2016-05-16Bibliographically 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.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 12:19
Keyword
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
Identifiers
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)
Opponent
Supervisors
Projects
The Human Protein Atlas
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20121017

Available from: 2012-10-17 Created: 2012-10-16 Last updated: 2013-04-15Bibliographically approved
2. Antibody-based subcellular localization of the human proteome
Open this publication in new window or tab >>Antibody-based subcellular localization of the human proteome
2016 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the use of antibodies and immunofluorescence for subcellular localization of proteins. The key objective is the creation of an open-source atlas with information on the subcellular location of every human protein. Knowledge of the spatial distribution and the precise location of a protein within a cell is important for its functional characterization, and describing the human proteome in terms of compartment proteomes is important to decipher cellular organization and function.

 

Immunofluorescence and confocal microscopy of cultured cells were used for high-resolution detection of proteins on a high-throughput scale. Critical to immunofluorescence results are sample preparation and specific antibodies. Antibody staining of cells requires fixation and permeabilization, both of which can result in loss or redistribution of proteins and masking of epitopes. A high-throughput approach demands a standardized protocol suitable for the majority of proteins across cellular compartments. Paper I presents an evaluation of sample preparation techniques from which such a single fixation and permeabilization protocol was optimized. Paper II describes the results from applying this protocol to 4000 human proteins in three cell lines of different origin.

 

Paper III presents a strategy for application-specific antibody validation. Antibodies are the key reagents in immunofluorescence, but all antibodies have potential for off-target binding and should be validated thoroughly. Antibody performance varies across sample types and applications due to the competition present and the effect of the sample preparation on antigen accessibility. In this paper application-specific validation for immunofluorescence was conducted using colocalization with fluorescently tagged protein in transgenic cell lines. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. viii, 53 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:13
Keyword
Human proteome, Subcellular localization, Organelles, Immunofluorescence, Fixation, Permeabilization, Antibody validation
National Category
Cell Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-186138 (URN)978-91-7729-010-0 (ISBN)
Presentation
2016-06-08, Alfa2, Tomtebodavägen 23A, Solna, 14:00 (English)
Opponent
Supervisors
Funder
Knut and Alice Wallenberg Foundation
Note

QC 20160509

Available from: 2016-05-16 Created: 2016-05-02 Last updated: 2016-05-16Bibliographically approved

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