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An antibody validation scheme for immunofluorescence using gene tagging
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. Science for Life Laboratory / Royal Institute of Technology. (Cell Profiling)ORCID iD: 0000-0002-2998-3077
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-186540OAI: oai:DiVA.org:kth-186540DiVA: diva2:927702
Note

QC 20160518

Available from: 2016-05-12 Created: 2016-05-12 Last updated: 2016-05-18Bibliographically approved
In thesis
1. 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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