Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy
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 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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- "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 . 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 –. 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. "
ABSTRACT: Affinity proteins binding to antibody constant regions have proved to be invaluable tools in biotechnology. Here, protein engineering was used to expand the repertoire of available immunoglobulin binding proteins via improvement of the binding strength between the widely used staphylococcal protein A-derived Z domain and the important immunoglobulin isotype mouse IgG(1) (mIgG(1)). Addressing seven positions in the 58-residue three-helix bundle Z domain by single or double amino acid substitutions, a total of 170 variants were individually constructed, produced in E. coli and tested for binding to a set of mouse IgG(1) monoclonal antibodies (mAbs). The best variant, denoted Z(F5I) corresponding to a Phe to Ile substitution at position 5, showed a typical ten-fold higher affinity than the wild-type as determined by biosensor technology. Eight amino acid positions in the Z(F5I) variant were separately mutated to cysteine for incorporation of a photoactivable maleimide-benzophenone (MBP) group as a probe for site-specific photoconjugation to Fc of mIgG(1), The best photocoupling efficiency to mIgG(1) Fc was seen when the MBP group was coupled to Cys at position 32, resulting in adduct formation to more than 60% of all heavy chains, with no observable non-selective conjugation to the light chains. A similar coupling yield was obtained for a panel of 19 different mIgG(1) mAbs, indicating a general characteristic. To exemplify functionalization of a mIgG(1) antibody via site-specific biotinylation, the Z(F5I-Q32C-MBP) protein was first biotinylated using an amine reactive reagent and subsequently photoconjugated to an anti-human interferon-gamma mIgG(1) mAb. When comparing the specific antigen binding ability of the probe-biotinylated mAb to that of the directly biotinylated mAb, a significantly higher bioactivity was observed for the sample biotinylated using the Z(F5I-Q32C-MBP) probe. This result indicates that the use of a site-specific and affinity probe-mediated conjugation strategy can result in antibody reagents with increased assay sensitivity.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
Article: Bioanalysis of Eukaryotic Organelles[Show abstract] [Hide abstract]
ABSTRACT: The role that organelle analysis has played in understanding biology is studied. Organelle analysis enables a more specific description of the molecular, biochemical, and physiological processes associated with diseases, embryonic development, tissue differentiation, organism aging, disease treatments, and organism response to pathogens. Confocal microscopy has become a routine tool for investigating subcellular organization, organelle networks, and organelle dynamics in cellular and tissue samples. Most organelles have a dynamic, three-dimensional (3D) organization inside the cell, which is tightly connected to their physiological functions. Due to this, a single 2D image inherently limits the information acquired about the distribution of a particular property within the organelle. The combination of subcellular fractionation with 'omic' technologies has become a powerful resource to characterize and catalogue the various subcellular environments in a cell.Chemical Reviews 04/2013; 113(4):2733-811. DOI:10.1021/cr300354g · 46.57 Impact Factor