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Antibody-based subcellular localization of the human proteome
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. Science for Life Laboratory / Royal Institute of Technology. (Cell Profiling)ORCID iD: 0000-0002-2998-3077
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 [en]
Human proteome, Subcellular localization, Organelles, Immunofluorescence, Fixation, Permeabilization, Antibody validation
National Category
Cell Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-186138ISBN: 978-91-7729-010-0 (print)OAI: oai:DiVA.org:kth-186138DiVA: diva2:925665
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
List of papers
1. A single fixation protocol for proteome-wide immunofluorescence localization studies
Open this publication in new window or tab >>A single fixation protocol for proteome-wide immunofluorescence localization studies
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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.

Keyword
Antibody, Confocal microscopy, Fixation, Immunofluorescence, Organelle, Permeabilization
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-29841 (URN)10.1016/j.jprot.2009.10.012 (DOI)000277763800004 ()2-s2.0-77950516661 (Scopus ID)
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
2. Mapping the subcellular protein distribution in three human cell lines
Open this publication in new window or tab >>Mapping the subcellular protein distribution in three human cell lines
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2011 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, no 8, 3766-3777 p.Article in journal (Refereed) Published
Abstract [en]

The subcellular locations of proteins are closely related to their function and constitute an essential aspect for understanding the complex machinery of living cells. A systematic effort has been initiated to map the protein distribution in three functionally different cell lines with the aim to provide a subcellular localization index for at least one representative protein from all human protein-encoding genes. Here, we present the results of over 4,000 proteins mapped to 16 subcellular compartments. The results indicate a ubiquitous protein expression with a majority of the proteins found in all three cell lines and a large portion localized to two or more compartments. The inter-relationships between the subcellular compartments are visualized in a protein-compartment network based on all detected proteins. Hierarchical clustering was performed to determine how closely related the organelles are in terms of protein constituents and compare the proteins detected in each cell type. Our results show distinct organelle proteomes, well conserved across the cell types, and demonstrate that biochemically similar organelles are grouped together.

Keyword
antibody, organelle, Human Protein Atlas, subcellular atlas, immunofluorescence, confocal microscopy
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-31514 (URN)10.1021/pr200379a (DOI)000293487900041 ()2-s2.0-79961240625 (Scopus ID)
Funder
Knut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20110905

Available from: 2011-03-17 Created: 2011-03-17 Last updated: 2017-12-11Bibliographically approved
3. An antibody validation scheme for immunofluorescence using gene tagging
Open this publication in new window or tab >>An antibody validation scheme for immunofluorescence using gene tagging
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-186540 (URN)
Note

QC 20160518

Available from: 2016-05-12 Created: 2016-05-12 Last updated: 2016-05-18Bibliographically approved

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Skogs, Marie

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