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Toward a confocal subcellular atlas of the human proteome
KTH, Skolan för bioteknologi (BIO).
KTH, Skolan för bioteknologi (BIO), Proteomik.ORCID-id: 0000-0001-7034-0850
KTH, Skolan för bioteknologi (BIO), Proteomik.ORCID-id: 0000-0003-3014-5502
KTH, Skolan för bioteknologi (BIO).
Vise andre og tillknytning
2008 (engelsk)Inngår i: Molecular and cellular proteomics, ISSN 1535-9476, Vol. 7, nr 3, s. 499-508Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

sted, utgiver, år, opplag, sider
2008. Vol. 7, nr 3, s. 499-508
Emneord [en]
HIGH-THROUGHPUT MICROSCOPY; FLUORESCENCE MICROSCOPY; AUTOMATED MICROSCOPY; MEMBRANE-PROTEINS; LOCALIZATION; CELLS; TISSUES
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-4882DOI: 10.1074/mcp.M700325-MCP200ISI: 000254076400004Scopus ID: 2-s2.0-41549148628OAI: oai:DiVA.org:kth-4882DiVA, id: diva2:1754
Merknad
QC 20100824Tilgjengelig fra: 2008-09-16 Laget: 2008-09-16 Sist oppdatert: 2020-01-10bibliografisk kontrollert
Inngår i avhandling
1. Bioimaging for analysis of protein expression in cells and tissues using affinity reagents
Åpne denne publikasjonen i ny fane eller vindu >>Bioimaging for analysis of protein expression in cells and tissues using affinity reagents
2008 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

The detection and analysis of biomolecules, such as proteins, are of great interest since these molecules are fundamental for life and our health. Due to the complexity of biological processes, there is a great advantage of studying proteins in their natural context, for example by using bioimaging. The objective of this doctoral thesis has been to develop, implement and evaluate techniques for the use of proteinspecific affinity reagents in diverse bioimaging platforms for analysis of protein expression in situ in cells and tissues.

To be able to visualize a desired protein in situ using affinity reagents, reporter labels are needed. A novel technique for labeling of antibodies on solid phase was developed. This method offers simultaneous purification, concentration and labeling of an antibody sample, giving highly predictable and reproducible results, in a miniaturized format.

Another study demonstrates the use of an alternative affinity reagent, the Affibody molecule, in bioimaging as well as other immunoassays. As a relevant proof-of-principle, an Affibody molecule binding the HER2 receptor was site-specificly labeled and employed for analysis of HER2 protein expression in cells and tissue using immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation and flow cytometry.

Furthermore, it is shown how antibody-based bioimaging approaches can be applied for systematic analysis of protein expression in terms of subcellular localization and expression levels in cell lines. The systematic subcellular localization of nearly 500 proteins was performed using IF and confocal microscopy. Global analysis of expression levels of nearly 2000 proteins in a panel of cell lines using IHC and automated image analysis, revealed that most proteins are expressed in a cell size dependent manner. Two normalization approaches were evaluated and found to allow for protein profiling across the panel of morphologically diverse cells, revealing patterns of protein over- and underexpression, and proteins with stable as well as with lineage specific expression were identified.

Finally, the value of antibody-based, bioimaging proteomics as a platform for biomarker discovery is demonstrated. The identification and in depth study of a candidate biomarker for colorectal cancer, SATB2, is described using both IHC and IF bioimaging. Results from extended analyses of tumor biopsies showed that detection of SATB2 protein using IHC provides a clinically relevant diagnostic tool with high specificity and sensitivity to aid in diagnosis of colorectal cancer. Furthermore, the study demonstrated a potential prognostic role of SATB2, as decreased expression was associated with a significantly shorter overall survival in patients with advanced colorectal cancer.

sted, utgiver, år, opplag, sider
Stockholm: KTH, 2008. s. vii, 86
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2008:14
Emneord
Affibody, antibody, biomarker, cell line, cell microarray, colorectal cancer, confocal microscopy, HER2, immunohistochemistry, immunofluorescence, light microscopy, protein expression, protein localization, SATB2, tissue microarray
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-4862 (URN)978-91-7415-096-4 (ISBN)
Disputas
2008-09-26, FD5, AlbaNova Universitetscentrum, Kungl Tekniska Högskolan, Roslagstullsbacken 21, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad
QC 20100824Tilgjengelig fra: 2008-09-05 Laget: 2008-09-05 Sist oppdatert: 2010-08-24bibliografisk kontrollert

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