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Antibody validation in bioimaging applications based on endogenous expression of tagged proteins Marie Skogs, Charlotte Stadler, Rutger Schutten, Martin Hjelmare, Christian Gnann, Lars Björk, Ina Poser, Anthony A. Hyman, Mathias Uhlén, and Emma K. Lundberg J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00821 • Publication Date (Web): 11 Oct 2016 Downloaded from http://pubs.acs.org on October 19, 2016

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Journal of Proteome Research

Antibody validation in bioimaging applications based on endogenous expression of tagged proteins

Marie Skogs1, Charlotte Stadler1, Rutger Schutten1, Martin Hjelmare1, Christian Gnann1, Lars Björk1, Ina Poser2, Anthony Hyman2, Mathias Uhlén1, Emma Lundberg1*

1. Science for Life Laboratory, Royal Institute of Technology, Stockholm, Sweden 2. Max Planck Institute, Molecular Cell Biology and Genetics, Dresden, Germany

* Corresponding Author: Correspondence should be addressed to Emma Lundberg ([email protected])

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ABSTRACT: 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 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 immunofluorescence applications using endogenous expression of tagged proteins. This is an important step towards a reproducible approach for context and application specific antibody validation and improved reliability of antibody-based experiments and research data.

Keywords: antibody validation, spatial proteomics, GFP, Human Protein Atlas, subcellular localization, immunofluorescence, confocal microscopy


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INTRODUCTION Recent discussions regarding reproducibility issues in life science research have highlighted problematic aspects surrounding the varying quality and consistency of antibodies for basic and translational research1,2. Several studies have reported that up to half of commercially available antibodies have problems with cross-reactivity or in some cases cannot even detect the target protein at endogenous levels3-6. To draw valid conclusions from any experiment one must be confident with the quality of the reagents used. Although antibodies have been used as indispensible research tools for many years, the scientific community has not adopted standardized procedures to validate their specificity. Several strategies have been suggested on how to improve this. Some researchers suggest that the problem is best tackled by only using recombinant antibodies with known sequence as this would be the most standardized and reproducible source7. Others recognize the continuous need for polyclonal antibodies due to the lower cost and better performance across multiple applications8, and consider the key issue to be improved validation procedures for identification of high quality antibodies9. The importance of a correct and detailed system for referencing of antibodies has also been acknowledged as an important factor for improving reproducibility10,11. Recently an international working group of scientist suggested five scientific principles for antibody validation in an applicationspecific manner12. It is critical that the antibody is validated for the intended application since antibody performance often differs across applications. Both specificity and cross-reactivity is highly dependent on the concentration of the target relative to competing off-target proteins13 and in a biological sample there are plenty of competing epitopes. Investigation of application specific antibody validation has been performed for many methods such as Western Blot13 and Immunoprecipitation14. In immunocytochemistry with immunofluorescence (IF) the choice of fixation and permeabilization protocol can influence antibody performance by changing epitope accessibility or by extraction of target proteins 15-17. Validation for IF should therefore be performed using the intended sample preparation protocol. Silencing or knockdown of the target with siRNA or CRISPR/Cas91,18,19, or the use of paired antibodies, two or more antibodies generated towards different parts of the same protein, have been demonstrated as useful validation strategies for IF20. The Human Protein Atlas (HPA) project is an effort aimed at antibody-based spatial characterization of the human proteome across a multitude of human tissues and cells20. For this purpose, rabbit polyclonal antibodies, targeting the majority of all human proteins have been produced. Part of this work is the subcellular localization of proteins using IF and confocal microscopy. The antigens used for antibody production are carefully selected to minimize crossreactivity21 and the antibodies are affinity purified using the antigen as affinity ligand22 and validated for reactivity to the antigen over a background of other antigens on protein arrays22.



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In this study, we describe a new approach for antibody validation based on detection of fluorescently tagged target protein expressed at endogenous levels in transgenic cell lines. We designed a systematic scheme for assessing antibody on- and off- target binding in IF applications and used it to investigate the performance of 197 HPA antibodies. The capability to bind also the endogenous non-tagged protein was subsequently investigated for antibodies successfully binding the fluorescently tagged target and observed discrepancies were examined using gene silencing. The antibody performance in IF was compared with the performance in Western Blot. Finally, polyclonal batch-to-batch variations were assessed for 35 antibodies where a second batch of antibody was produced by immunization of the same antigen in another rabbit. We conclude that expression of a tagged protein at endogenous levels is an attractive path for systematic identification and validation of antibodies in bioimaging applications.

MATERIALS AND METHODS Human Protein Atlas antibodies Rabbit polyclonal antibodies were generated within the HPA project towards antigens of 100-150 amino acids, called Protein Epitope Signature Tags (PrESTs), selected using an in house program to minimize cross-reactivity21. The antibodies were affinity purified using the antigen as affinity ligand23 and specificity was validated on protein arrays as previously described 22,24. Fluorescent protein tagging HeLa-Kyoto cell lines stably expressing the target protein tagged with Enhanced Green Fluorescent Protein (EGFP) were produced as previously described22 by the group of Anthony Hyman, Max Planck Institute Dresden, Germany. In brief Bacterial Artificial Chromosomes (BAC) were selected for each gene and tagged at either N- or C-terminal with EGFP combined with purification and cleavage tags through recombineering in 96-well format in E.coli followed by stable transfection into HeLa-Kyoto cells. All analyses were performed on a cell pool with different copy number and integration site of the transgene. Cell line cultivation HeLa (ACC 57, DSMZ, Germany) and BAC-transgenic HeLa-Kyoto (a gift from Anthony Hyman, Max Planck Institute, Dresden, Germany) were cultured in Dulbecco's Modified Eagle Medium and U-2 OS (LGC/ATCC, US) supplemented with 10% fetal bovine serum at 37°C, 5% CO2 and humidified air. For transgenic cells 0.8 % Geneticin (Sigma-Aldrich, US) was added to the media to maintain transgene expression in the cell population. Untransfected HeLa and a selection of transgenic cell lines were tested negative for mycoplasma using Venor GeM Advance (Minerva Biolabs, Germany). RNA sequencing for HeLa and U-2 OS were previously performed as a part of the Human Protein Atlas25.


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Immunofluorescence Immunofluorescent stainings were performed in 96-well glass bottom plates (Greiner, Kremsmuenster, Austria) using a Tecan Freedom EVO 150 liquid handling system (Tecan, Switzerland). Wells were coated with human fibronectin (VWR, US) at 12.5 µg/ml in PBS in 4°C overnight. 8000 cells were seeded per well and allowed to attach overnight followed by fixation with 4% paraformaldehyde in complete growth medium for 15 minutes and permeabilization with 0.1% Triton X-100 in PBS for 3x5 minutes (Sigma-Aldrich, US). Cells were incubated with primary antibodies in PBS with 4% FBS overnight, washed in PBS and incubated with secondary antibodies in 4% FBS for 1.5 hours. After washing with PBS cells were counterstained with 4’,6Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (LifeTechnologies, US) diluted to 300 nM in PBS for 10 minutes. Cells were once again washed and wells were mounted with PBS with 78% glycerol and sealed. Three different stainings were performed in parallel for each antibody-transgenic cell line pair; one well with transgenic cell line to examine tagged protein location, one well with HPA antibody in transgenic cell line to assess overlap and one well with HPA antibody in un-transfected HeLa. Detection of EGFP was enhanced by a mouse monoclonal antibody to GFP diluted 1:1000 (ab1218, RRID:AB_298911, Abcam, UK). HPA antibodies were used at 2-4 µg/ml. Cell quality was visualized by detecting tubulin with a rat monoclonal antibody diluted 1:2500 (ab6160, RRID:AB_305328, Abcam, UK). All secondary antibodies were Alexa coupled goat antibodies diluted 1:800; 488-anti-mouse, 555-anti-rat and 647-anti-mouse (A11029 RRID:AB_10566286, A21434 RRID:AB_10562898, A21245 RRID:AB_10562892, Life Technologies, USA). Image acquisition Confocal images were manually acquired using a Leica SP5 laser scanning confocal microscope (DM6000CS) equipped with a 63x HCX PL APO 1.40 oil CS objective (Leica Microsystems, Mannheim, Germany). The microscope software used was LAS AF (LAS AF 2.6.0 BETA build 6964, Leica Microsystems). Images were acquired in four sequential steps with the following scanning settings: 16 bit, 600 Hz, line average 2, pixel size 0.08 μm. Images were colored and assembled as RGB with the software ImageJ 1.44o (National Institutes of Health, USA). Several images were acquired with different detector gain settings to capture the heterogeneity within the EGFP-expressing cell population. Annotation All images were manually annotated to one or several of 22 subcellular locations in combination with one or several of 7 staining characteristics, if applicable. For the experiment with antibody staining in transgenic cells the overlap of antibody staining and tagged protein was manually assessed and scored as either Supportive: Antibody staining overlaps with tagged protein/Antibody staining overlaps with tagged protein but show additional locations, Uncertain: No antibody staining/No tagged protein/No antibody staining or tagged protein or Fail: Antibody staining does not bind tagged protein. To examine the complementarity of antibody based localization with protein tagging the locations obtained with antibodies in untransfected HeLa cells were compared to



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the locations seen for the tagged protein and scored as: Tagged protein and antibody staining show identical results, Tagged protein shows additional locations, Antibody staining shows additional locations, Tagged protein and antibody staining both show additional locations, Tagged protein and antibody staining have no overlapping locations or No staining in untransfected cells. siRNA knockdown A 96-well glass bottom plate (Greiner, Kremsmuenster, Austria) was coated with human fibronection (VWR, US) at 12.5 µg/ml in PBS in 4°C overnight. 5000 HeLa cells or 8000 U-2 OS cells were seeded the day prior to transfection in complete growth media. Target siRNA (TOMM40 s20448 and RNF2 s12067, LifeTechnologies) or scrambled siRNA (Silencer Select negative control 1, 4390844, ThermoFisher) were diluted in RNase free water to a 4µM stock solution. 50 µl Opti-MEM (31985-062, ThermoFisher), 0.5 µl siRNA stock solution and 1 µl Interferin (409-10, ThermoFisher) were mixed and incubated for 10 minutes. Transfection mix was added to the cells together with 150 µl fresh complete growth media for a siRNA working concentration of 10nM. After 24 hours the media was exchanged and after 48 hours the cells were fixed, permeabilized and stained.

RESULTS Design of antibody validation scheme using gene-tagging Here we present a strategy to systematically validate antibodies for IF using gene tagging as outlined in Figure 1. The first part of the validation scheme aims to identify the antibodies with capability to detect the target protein. In this step, antibodies were used to stain transgenic cells expressing the corresponding target protein fused to a green fluorescent protein (EGFP). The on- and off-target binding capabilities were assessed by evaluating the co-localization between the EGFP- and antibody signal. In another set of experiments we aim to validate if the antibodies also can bind the endogenous protein. In this step, the antibodies capable of detecting the tagged protein were used to stain the endogenous protein in non-transfected cells.


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Antibodies were analyzed in transgenic cell lines expressing a EGFP tag to the target protein at near endogenous levels.

Target

No - antibody staining does not overlap with tagged protein.

EGFP

Antibodies were analyzed in untransfected cells expressing the endogenous protein only.

Yes - antibody binds tagged protein. Maybe - IF of endogenous protein show dissimilar location(s) to the tagged protein.

Yes - IF of endogenous protein show identical location(s) to the tagged protein.

Figure 1. Overview of antibody validation scheme. In step 1, antibodies were used to stain transgenic cells expressing tagged target protein. In step 2, antibodies capable of detecting the tagged protein were used to stain the endogenous protein in untransfected cells and the obtained locations for tagged protein and endogenous protein were compared.

The antibodies used were 197 affinity-purified rabbit polyclonal antibodies generated within the HPA project (Supplementary Table 1). The transgenic cell lines used were HeLa cells transfected with bacterial artificial chromosomes (BAC)26 (Supplementary Table 2). In contrast to traditional fluorescent protein tagging based on cDNA and standard promotors that often lead to ectopic overexpression and potential mislocalization of the target protein27,28, the large transgenic inserts based on BACs results in a near endogenous expression as the transgene contains the tagged protein in its native context with native promoter, upstream and downstream regulatory elements as well as intron structure. Only tagged proteins showing the expected cellular locations according to UniProt were employed in this study. Detection of tagged protein The 108 transgenic cell lines used were each stained with 1-3 antibodies generated towards different epitopes on the wild-type target protein. Within the HPA project there were a total of 197 antibodies against these proteins that had passed our quality control22. These were subsequently used to stain the corresponding transgenic cell lines expressing the target protein tagged with EGFP at either the N- or C terminus. Cells were fixed, permeabilized and stained with an antibody for the target protein as well as an anti-GFP antibody in order to detect even low abundant tagged proteins. We used the same automated sample preparation protocol that is optimized for high-throughput IF in the HPA project16. The analysis was performed on a pool of transfected cells with average expression at endogenous levels in bulk measurements29. A variation could be observed in the expression level of tagged protein per cell, and the fraction of cells with any detectable tagged protein varied (e.g. DAP3 in Figure 3). In



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addition to the EGFP-tagged protein, the transgenic cells also express the endogenous protein that was also detected by the antibody.

Figure 2. Overview of the results obtained in step 1 and step 2. Number of antibodies in parenthesis, total number of antibodies in step 1 was 197 and in step 2 136. The number of antibodies that are potentially good for IF but is suggested for further validations are seen in the yellow, orange and sepia colored section (N=6, 44, 10 respectively). Of the 197 antibodies analyzed, 69% showed a staining that overlapped with the tagged protein (Fig 2). Examples are antibody HPA026111 targeting the intermediate filament protein Nestin (NES) and HPA044581 targeting VPS26 retromer complex component A (VPS26A) that transports proteins from endosomes to the trans-Golgi network (Fig. 3). 23% of the antibodies gave a staining that did not overlap with the tagged protein and 8% did not give any staining at all. An example of a non-successful validation is HPA071591, raised towards another part of VPS26A that showed no overlap with the tagged protein and instead gave a nuclear pattern. Many of the antibodies with immunoreactivity overlapping the tagged protein also showed additional non-overlapping staining. Reasons for this could be either cross reactivity of the antibody, presence of endogenous non-tagged protein, or a location of the endogenous target not being accessible by the tagged protein. Examples are shown in Figure 3, the antibody HPA023687 targeting the mitochondrial protein Death associated protein 3 (DAP3) and HPA036231 targeting the mitochondrial protein Translocase of outer mitochondrial membrane 40 homolog (TOMM40). Both overlap with the tagged protein in mitochondria but also show additional locations; nucleus and cytoplasm respectively.


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Figure 3. IF images of colocalization with tagged protein and staining of endogenous protein.



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IF stainings of tagged protein in transgenic cell lines and endogenous protein in untransfected cells for selected antibodies. Nuclear marker DAPI in blue included for selected stainings. Scale bar indicates 30µm.

Detection of endogenous protein To validate whether the antibodies could detect the endogenous protein, the 136 antibodies capable of binding the tagged protein were used to stain untransfected HeLa cells. Results are shown in Figure 2 and Supplementary Table 1. For 53 of these antibodies the obtained locations were identical to the tagged protein. Good examples are NES and VPS26A as shown in Figure 3. The majority of the stainings performed displayed the same location(s) of the tagged protein and the endogenous protein, but additional locations were also observed for either the endogenous protein (n = 44), the tagged proteins (n = 6) or both (n=10). Examples are TOMM40 (Fig. 3) where the tagged protein localizes to mitochondria whereas the endogenous protein is detected also in cytoplasm, and Nuclear assembly factor 1 ribonucleoprotein (NAF1) where the tagged protein localizes to nucleus and cytoplasm while the endogenous protein is only detected in the nucleus. For 11 of the antibodies there were no shared locations between tagged and endogenous protein, as illustrated by TERF1-interacting nuclear factor 2 (TINF2) (Fig. 3) where tagged protein localizes to spots in the nucleus but the endogenous protein is detected in endoplasmic reticulum. The RNA levels for all target genes were analyzed by RNA-Seq and detectable levels of transcripts (FPKM >3 which is Fragments Per Kilobase of exon per Million fragments mapped25) were found for all 136 genes capable of binding tagged protein (Supplementary Table 1). The groups where an additional location was seen for the tagged protein or where no staining at all was detected for the endogenous protein by the antibody, also had the lowest average expression at RNA-level based on FPKM-values. This indicates that the expression levels in non-transfected HeLa cells were not high enough to enable detection without the addition of tagged protein.

Validation of discrepancies using gene silencing To investigate whether differences in antibody staining of tagged protein and endogenous protein were due to cross-reactivity of the antibody or represented true protein detection we performed additional antibody validation by siRNA gene silencing for two genes. For the mitochondrial protein TOMM40 shown in Figure 3, the tagged protein localized to mitochondria whereas the antibody HPA036231 stained both mitochondria and cytoplasm. Staining with this antibody in other cell lines such as U-2 OS cells also showed mitochondria and cytoplasm (Supplementary Fig. 1). Silencing of the target protein in HeLa cells led to loss of the mitochondrial stain but not the cytoplasmic, indicating that the cytoplasmic staining is due to cross-reactivity (Fig. 4). FPKM value for HeLa cells were 25 and 60 for U-2 OS. For TINF2 in Figure 3, the tagged protein localized to distinct spots in the nucleus. The antibody HPA059061 showed a perfect overlap in the nuclear spots in transgenic cells, but staining of endogenous protein in non-transfected cells showed endoplasmic reticulum. Staining in U-2 OS cells also displayed nuclear spots (Fig. 4) and knockdown of the target protein in HeLa and U-2 OS cells led to


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loss of the nuclear spots in U-2 OS but not the endoplasmic reticulum in HeLa (Fig. 4). This shows that the antibody targets the correct protein, but also has offtarget binding activity localized to the ER in non-tranfected HeLa cells. The FPKM value was 15 for HeLa cells and 19 for U-2 OS. These results emphasize the need for antibody validation in the context of intended use, both in terms of sample preparation, application and choice of model system (cell line).

Figure 4. siRNA knockdown. Knockdown of TOMM40 leads to loss of mitochondrial staining but not cytoplasmic. Knockdown of TINF2 leads to loss of nuclear staining in U-2 OS but not of endoplasmic reticulum staining in HeLa. Antibody staining in green. Scale bar indicates 50 µm.



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Batch-to-batch variations of polyclonal antibodies 35 antibodies that were validated to detect the tagged protein, were investigated for batch-to-batch variations by analyzing a second batch of antibodies produced from immunization of the same antigen in another rabbit (Supplementary Table 3). These antibodies were also affinity purified using the antigen as affinity ligand. Of the new batches, 23 antibodies (66%) were again capable of detecting the tagged protein, while 12 (34%) failed to detect the tagged protein (Figure 5).

Figure 5. Batch-to-batch comparison For 35 antibodies capable of binding the tagged protein a second batch of polyclonal antibody was obtained and analyzed. Of the 23 new batches that did detect tagged protein only 7 showed an identical staining pattern to the first batch when analyzing endogenous protein. Figure 6 shows three pairs of antibody batches, where the first pair targeting vesicleassociated membrane protein-associated protein A (VAPA) both overlapped with tagged protein in endoplasmic reticulum and showed the same location in nontransfected cells. The two batches targeting RAD18 E3 ubiquitin protein ligase (RAD18) overlapped with tagged protein in nucleus but in non-transfected cells one of the batches showed an additional staining of the Golgi apparatus. The two batches targeting Zinc finger protein 687 (ZNF687) both detected the tagged protein in transgenic cells but HPA023948 failed to detect the endogenous target and HPA053052 showed a strong nucleoli staining. These results demonstrate that both the on- and off-target capabilities of antibodies can vary from batch to batch.


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Figure 6. IF stainings of batch-to-batch variations Batch-to-batch variations between antibody batches to three different antigens. Scale bar indicates 30µm



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Correlation between antibody performance in Western Blot and Immunofluorescence In order to investigate the application dependency of antibody performance, the antibodies used in this study were also analyzed by Western blot (WB) using the HPA standardized high-throughput setup13 using five different samples (Supplementary Table 1). Among the antibodies that could detect the tagged protein 47% detected a band of the expected size in WB (score 1 and 2 in Supplementary Table 1) compared to 39 % for the antibodies that did not detect the tagged protein and thereby were not qualified to be suitable for IF. There was thus low correlation between results in the two applications; hence the performance of antibodies in WB cannot be predicted by their performance in IF (Supplementary Figure 2). This is not surprising, since the target proteins have been treated considerably differently in the two applications, such as complete protein denaturation with SDS in Western blot and a milder cross-linking of the protein using paraformaldehyde in the bioimaging application.

DISCUSSION It is well known that the performance of antibodies is both application and context dependent. Several studies have reported that up to half of commercially available antibodies have problems with cross-reactivity or in some cases cannot even detect the target protein at endogenous levels3-6, yet the scientific community has not adopted standardized procedures for validation of antibodies in the context and application of its intended use. In this study we demonstrate the use of gene tagging as a method for systematic validation of antibodies suitable for IF applications. In a first step the capability of binding to the tagged-protein is tested. In a second step the capability of binding to the endogenous protein is tested. One major advantage of this approach is that it identifies antibodies capable of binding the target protein using the identical fixation, permeabilization and staining conditions of choice. The effect of antibody binding using different sample preparation protocols are easily tested and the best condition can be optimized. Potential off-target binding is also easily identified as non-overlapping staining. It is these strengths that make this method a great complement for the existing methods for antibody validation in IF such as paired antibodies or gene knockdown/silencing. As the performance of antibodies is dependent on the concentration of both target and off-target proteins, it is of outmost importance to express the tagged protein at endogenous levels. Caution must also be taken to avoid potential artifacts introduced by the tag itself, such as altered subcellular localization or affected protein activity. We showed that there are discrepancies between studying endogenous and tagged proteins, even when tagged proteins are expressed at endogenous levels under endogenous control. In difficult cases, it is therefore recommended to add complementary validation strategies, such as gene knockout or silencing using siRNA or CRISPR/Cas9.


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We observed variations in the number of cells with detectable tagged protein as well as the amount of tagged protein per cell for some cell lines. It has been shown by WB that the amount of tagged protein for BAC transfected cells is similar to or lower than the endogenous counterpart when looking at a batch of cells29, but our results show that there are greater variations in cell-to-cell levels of tagged proteins than for the corresponding endogenous proteins. It has been reported that the BAC transgene is inserted at single or low copy number26 so the differences in intensity may be due to insertion position effects. For validation of antibodies in IF applications this is actually an advantage as variation in tagged protein levels between cells allow a more robust colocalization analysis. Today, the most commonly used control for antibody specificity is WB showing that the antibody produces a clear band of the expected size. In this study we show that successful antibody validation in IF did not predict antibody performance in WB or vice versa. This proves that application-specific validation cannot be circumvented and results cannot be extrapolated to other applications. All antibodies used in this study were affinity-purified using the antigen as the affinity ligand and they all passed a quality control step with a peptide array screening. Still 31% were not able to detect the target protein in IF and 56% did not give a band of expected size in WB. This could either be due to differences between binding to the antigen and a native full-length protein or reflect the fraction of antibodies that can be expected to work for each application. Both IF and WB were performed with a standardized high-throughput protocol and it is reasonable to believe that testing different protocols, samples and conditions would result in a higher fraction of successful antibodies for each application. We furthermore investigated the antibody batch-to-batch effect in terms of onand off-target binding by assessing 35 antibodies and subsequently assess them using the developed scheme. Large differences could be observed between some of the antibody batches. About one third of re-immunizations failed to even produce antibodies capable of detecting the target. Even among the antibodies where both batches were capable of detecting the tagged protein, some differences in the observed staining pattern could be observed, e.g. additional weak off-target staining. Epitope mapping of HPA antibodies has shown that different batches have a similar but not identical epitope panels and also differences in the relative amount of antibodies towards the different epitopes30.

CONCLUSIONS These results illustrate the necessity for validation of every new antibody batch and the importance of including batch information when referencing antibodies. Based on the results of this study, the subcellular program of the Human Protein Atlas consortium will use the method described here as one of their validation “pillars” and the results from the corresponding antibodies will be presented as part of the validation pages in the Protein Atlas database.



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Despite the issues with varying quality and reproducibility, antibodies are invaluable tools for basic and translational life science research. Unfortunately there is seldom a perfect target-specific antibody functional in all applications. We suggest that antibodies, and other affinity reagents, should be viewed upon in a similar way as small compounds. Each reagent has the potential of both onand off-target binding and should be validated in a context and application specific manner. Here we present a scheme for antibody validation in IF applications using gene tagging as a step towards a reproducible approach for improved antibody validation and generation of reliable antibody data.

SUPPORTING INFORMATION The following files are available free of charge at the ACS website http://pubs.acs.org: Supplementary figures: Supplementary_figure_1.pdf Supplementary_figure_2.pdf Supplementary tables: Supplementary table 1.xls Supplementary table 2.xls Supplementary table 3.xls AUTHOR INFORMATION Corresponding Author * Correspondence should be addressed to Emma Lundberg ([email protected]) Author contributions M.S., C.S., R.S., C.G. and M.H performed the IF experiments. M.S performed siRNA experiments. I.P. and A.H. provided the transgenic cell lines. L.B and M.U provided intellectual input. E.L. designed and led the study. E.L., M.S. and C.S. wrote the manuscript. ACKNOWLEDGEMENTS We acknowledge the entire staff of the Human Protein Atlas program. Funding were provided by the Knut and Alice Wallenberg Foundation, the Erling Persson Foundation and facility support to the Science for Life Laboratory (SciLifeLab).


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Similar, but Not Identical, Epitopes. PLoS ONE 2012, 7 (12), e45817.



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Antibody Validation in Bioimaging Applications ... - ACS Publications

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