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Antibody Validation in Bioimaging Applications Based on Endogenous Expression of Tagged Proteins
KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-2998-3077
KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-8787-8868
KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-7375-9681
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2017 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 16, no 1, p. 147-155Article in journal (Refereed) Published
Abstract [en]

Antibodies are indispensible research tools, yet the scientific community has not adopted standardized procedures to validate their specificity. Here we present a strategy to systematically validate antibodies for immunofluorescence (IF) applications using gene tagging. We have assessed the on- and off-target binding capabilities of 197 antibodies using 108 cell lines expressing EGFP-tagged target proteins at endogenous levels. Furthermore, we assessed batch-to-batch effects for 35 target proteins, showing that both the on- and off-target binding patterns vary significantly between antibody batches and that the proposed strategy serves as a reliable procedure for ensuring reproducibility upon production of new antibody batches. In summary, we present a systematic scheme for antibody validation in IF applications using endogenous expression of tagged proteins. This is an important step toward a reproducible approach for context- and application-specific antibody validation and improved reliability of antibody-based experiments and research data.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2017. Vol. 16, no 1, p. 147-155
Keywords [en]
antibody validation, spatial proteomics, GFP, Human Protein Atlas, Cell Atlas, subcellular localization, immunofluorescence, confocal microscopy
National Category
Medical Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-201243DOI: 10.1021/acs.jproteome.6b00821ISI: 000391782100014PubMedID: 27723985Scopus ID: 2-s2.0-85017638855OAI: oai:DiVA.org:kth-201243DiVA, id: diva2:1074743
Note

QC 20170216

Available from: 2017-02-16 Created: 2017-02-16 Last updated: 2020-03-09Bibliographically approved

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Skogs, MarieStadler, CharlotteSchutten, RutgerHjelmare, MartinGnann, ChristianBjörk, LarsUhlén, MathiasLundberg, Emma
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