Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy

Journal of Proteomics
Charlotte StadlerEmma Lundberg

Abstract

We have developed a platform for validation of antibody binding and protein subcellular localization data obtained from immunofluorescence using siRNA technology combined with automated confocal microscopy and image analysis. By combining the siRNA technology with automated sample preparation, automated imaging and quantitative image analysis, a high-throughput assay has been set-up to enable confirmation of accurate protein binding and localization in a systematic manner. Here, we describe the analysis and validation of the subcellular location of 65 human proteins, targeted by 75 antibodies and silenced by 130 siRNAs. A large fraction of (80%) the subcellular locations, including locations of several previously uncharacterized proteins, could be confirmed by the significant down-regulation of the antibody signal after the siRNA silencing. A quantitative analysis was set-up using automated image analysis to facilitate studies of targets found in more than one compartment. The results obtained using the platform demonstrate that siRNA silencing in combination with quantitative image analysis of antibody signals in different compartments of the cells is an attractive approach for ensuring accurate protein localization as well as...Continue Reading

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Citations

Mar 4, 2014·International Immunopharmacology·Christina KalderénStefan Svensson Gelius
Mar 4, 2014·Journal of Proteome Research·Charlotte StadlerMathias Uhlén
Apr 11, 2013·Chemical Reviews·Chad P SatoriEdgar A Arriaga
Sep 7, 2016·Nature Methods·Mathias UhlenTadashi Yamamoto
Sep 25, 2016·Journal of Immunological Methods·Anastasia ZotovaDmitriy Mazurov
Oct 12, 2016·Journal of Proteome Research·Marie SkogsEmma Lundberg
May 13, 2017·Science·Peter J ThulEmma Lundberg
Sep 25, 2017·Protein Science : a Publication of the Protein Society·Peter J Thul, Cecilia Lindskog
Dec 19, 2019·Frontiers in Immunology·Rubin Narayan JoshiMattias Vesterlund
Jan 20, 2019·Nature Reviews. Molecular Cell Biology·Emma Lundberg, Georg H H Borner
Apr 3, 2020·Nature Protocols·James JonkmanAlison J North
Nov 5, 2020·Protein Science : a Publication of the Protein Society·Andreas Digre, Cecilia Lindskog
Jan 14, 2020·Hearing Research·Neil J InghamKaren P Steel

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