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

Science for Life Laboratory, Royal Institute of Technology, Stockholm, SE-171 65, Sweden.
Journal of proteomics (Impact Factor: 3.89). 02/2012; 75(7):2236-51. DOI: 10.1016/j.jprot.2012.01.030
Source: PubMed


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 antibody binding using immunofluorescence. With a large fraction of the human proteome still unexplored, we suggest this approach to be of great importance under the continued work of mapping the human proteome on a subcellular level.

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    • "This process makes it possible to produce selective polyclonal antibodies with low off-target affinities [11] [12] [13]. Within the HPA, all the antibodies produced are validated using multiple approaches to ensure that they bind to their intended target [11] [14]. One of these validation approaches involves the protein fragment microarrays, which are used for generating multiplex binding profiles of the antibodies [11]. "
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    ABSTRACT: High-density protein microarrays of recombinant human protein fragments, representing 12,412 unique Ensembl Gene IDs, have here been produced and explored. These protein microarrays were used to analyse antibody off-target interactions, as well as for profiling the human autoantibody repertoire in plasma against the antigens represented by the protein fragments. Affinity-purified polyclonal antibodies produced within the Human Protein Atlas (HPA) were analysed on microarrays of three different sizes, ranging from 384 antigens to 21,120 antigens, for evaluation of the antibody validation criteria in the HPA. Plasma samples from secondary progressive multiple sclerosis patients were also screened in order to explore the feasibility of these arrays for broad-scale profiling of autoantibody reactivity. Furthermore, analysis on these near proteome-wide microarrays was complemented with analysis on HuProt™ Human Proteome protein microarrays. The HPA recombinant protein microarray with 21,120 antigens and the HuProt™ Human Proteome protein microarray are currently the largest protein microarray platforms available to date. The results on these arrays show that the Human Protein Atlas antibodies have few off-target interactions if the antibody validation criteria are kept stringent and demonstrate that the HPA-produced high-density recombinant protein fragment microarrays allow for a high-throughput analysis of plasma for identification of possible autoantibody targets in the context of various autoimmune conditions.
    New Biotechnology 09/2015; DOI:10.1016/j.nbt.2015.09.002 · 2.90 Impact Factor
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    • "Antibodies play crucial roles in in vitro diagnostic assays of different complexity, involving either the use of single antibody reagents, or more commonly, by combining several antibodies in "sandwich" assays or more sophisticated set-ups [1]. In most cases, at least one of the antibodies used in the assay needs to be labeled allowing for direct or indirect detection via fluorescence, enzymatic conversion of suitable substrates, or DNA amplification/replication [2]–[4]. Such labeling of native antibody proteins is typically performed in a more or less uncontrolled manner in respect to the number and location of sites being modified. "
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    PLoS ONE 02/2013; 8(2):e56597. DOI:10.1371/journal.pone.0056597 · 3.23 Impact Factor
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    ABSTRACT: Imaging techniques such as immunofluorescence (IF) and the expression of fluorescent protein (FP) fusions are widely used to investigate the subcellular distribution of proteins. Here we report a systematic analysis of >500 human proteins comparing the localizations obtained in live versus fixed cells using FPs and IF, respectively. We identify systematic discrepancies between IF and FPs as well as between FP tagging at the N and C termini. The analysis shows that for 80% of the proteins, IF and FPs yield the same subcellular distribution, and the locations of 250 previously unlocalized proteins were determined by the overlap between the two methods. Approximately 60% of proteins localize to multiple organelles for both methods, indicating a complex subcellular protein organization. These results show that both IF and FP tagging are reliable techniques and demonstrate the usefulness of an integrative approach for a complete investigation of the subcellular human proteome.
    Nature Methods 02/2013; 10(4). DOI:10.1038/nmeth.2377 · 32.07 Impact Factor
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