A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence

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McPherson" }, { "@type": "Person", "name": "Carl Laflamme" }, { "@type": "Person", "name": "NeuroSGC/YCharOS/EDDU collaborative group" }, { "@type": "Person", "name": "ABIF consortium" } ], "publisher": { "@type": "Organization", "name": "F1000Research", "logo": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 480, "width": 60 } }, "image": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 1200, "width": 150 }, "description": "VAPB is an adaptor protein known for its role as an anchor for other proteins at the endoplasmic reticulum. A mutant form of VAPB has been linked to amyotrophic lateral sclerosis and the underlying mechanisms resulting from this defect are studied by researchers in this area to uncover its implication in the disease. Here we have characterized six VAPB commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs." } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/13-1559", "name": "A guide to selecting high-performing antibodies for VAPB (UniProt..." } } ] } Home Browse A guide to selecting high-performing antibodies for VAPB (UniProt... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article González Bolívar S, Ayoubi R, Alende C et al. A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 3 approved, 1 approved with reservations] . F1000Research 2024, 13 :1559 ( https://doi.org/10.12688/f1000research.160226.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Data Note A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 3 approved, 1 approved with reservations] Sara González Bolívar https://orcid.org/0000-0002-4299-8281 1 , Riham Ayoubi 1 , Charles Alende https://orcid.org/0009-0005-4611-6134 1 , [...] Maryam Fothouhi 1 , Irina Shlaifer 1 , Peter S. McPherson 1 , Carl Laflamme https://orcid.org/0000-0001-5906-025X 1 , NeuroSGC/YCharOS/EDDU collaborative group , ABIF consortium Sara González Bolívar https://orcid.org/0000-0002-4299-8281 1 , Riham Ayoubi 1 , [...] Charles Alende https://orcid.org/0009-0005-4611-6134 1 , Maryam Fothouhi 1 , Irina Shlaifer 1 , Peter S. McPherson 1 , Carl Laflamme https://orcid.org/0000-0001-5906-025X 1 , NeuroSGC/YCharOS/EDDU collaborative group , ABIF consortium PUBLISHED 24 Dec 2024 Author details Author details 1 Montreal Neurological Institute-Hospital, Montreal, Québec, Canada Sara González Bolívar Roles: Investigation Riham Ayoubi Roles: Investigation, Supervision, Writing – Original Draft Preparation Charles Alende Roles: Investigation Maryam Fothouhi Roles: Investigation Irina Shlaifer Roles: Investigation Peter S. McPherson Roles: Funding Acquisition Carl Laflamme Roles: Conceptualization, Funding Acquisition, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the YCharOS (Antibody Characterization through Open Science) gateway. Abstract VAPB is an adaptor protein known for its role as an anchor for other proteins at the endoplasmic reticulum. A mutant form of VAPB has been linked to amyotrophic lateral sclerosis and the underlying mechanisms resulting from this defect are studied by researchers in this area to uncover its implication in the disease. Here we have characterized six VAPB commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs. READ ALL READ LESS Keywords O95292, VAPB, Vesicle-associated membrane protein-associated protein B/C, VAMP-associated protein B/C, antibody characterization, antibody validation, western blot, immunoprecipitation, immunofluorescence Corresponding Author(s) Carl Laflamme ( [email protected] ) Close Corresponding author: Carl Laflamme Competing interests: For this project, the authors developed partnerships with high-quality antibody manufacturers and KO cell line providers. The partners provide antibodies and KO cell lines to the McPherson laboratory at no cost. These partners include: - Abcam-ABCD antibodies- ABclonal- Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific. Grant information: This work was supported by a grant from the Motor Neurone Disease Association (UK), The ALS Association (USA) and ALS Canada, by a Canadian Institutes of Health Research Foundation Grant (FDN154305) and by the Government of Canada through Genome Canada and Ontario Genomics (OGI-210). RA is supported by a Mitacs fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2024 González Bolívar S et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: González Bolívar S, Ayoubi R, Alende C et al. A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 3 approved, 1 approved with reservations] . F1000Research 2024, 13 :1559 ( https://doi.org/10.12688/f1000research.160226.1 ) First published: 24 Dec 2024, 13 :1559 ( https://doi.org/10.12688/f1000research.160226.1 ) Latest published: 24 Dec 2024, 13 :1559 ( https://doi.org/10.12688/f1000research.160226.1 ) Introduction VAPB is the vesicle-associated membrane protein (VAMP)-associated protein B and C. 1 VAPB acts as a tethering protein of the endoplasmic reticulum to other organelles, orchestrating many cellular processes, namely vesicle trafficking. 2 A mutant form of the protein has been linked to amyotrophic lateral sclerosis 3 and high-quality antibodies and necessary to explore the resulting pathological mechanisms. This research is part of a broader collaborative initiative in which academics, funders and commercial antibody manufacturers are working together to address antibody reproducibility issues by characterizing commercial antibodies for human proteins using standardized protocols, and openly sharing the data. 4 – 6 Here we evaluated the performance of six commercial antibodies for VAPB for use in western blot, immunoprecipitation, and immunofluorescence, enabling biochemical and cellular assessment of VAPB properties and function. The platform for antibody characterization used to carry out this study was endorsed by a committee of industry academic representatives. It consists of identifying human cell lines with adequate target protein expression and the development/contribution of equivalent knockout (KO) cell lines, followed by antibody characterization procedures using most commercially available antibodies against the corresponding protein. The standardized consensus antibody characterization protocols are openly available on Protocol Exchange, a preprint server (DOI: 10.21203/rs.3.pex-2607/v1 ). 7 The authors do not engage in result analysis or offer explicit antibody recommendations. Our primary aim is to deliver top-tier data to the scientific community, grounded in Open Science principles. This empowers experts to interpret the characterization data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs. Guidelines on how to interpret antibody characterization data found in this study are featured on the YCharOS gateway. 8 Results and discussion Our standard protocol involves comparing readouts from WT (wild type) and KO (knockout cells). 9 , 10 The first step was to identify a cell line(s) that expresses sufficient levels of a given protein to generate a measurable signal using antibodies. To this end, we examined the DepMap (Cancer Dependency Map Portal, RRID:SCR_017655) transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log 2 (transcripts per million “TPM” + 1), which we have found to be a suitable cut-off. 4 HeLa expresses the VAPB transcript at 5.2 and was identified as a suitable cell line to modify with CRISPR/Cas9 to KO the corresponding VAPB gene ( Table 1 ). Table 1. Summary of the cell lines used. Institution Catalog number RRID (Cellosaurus) Cell line Genotype ATCC CCL-2 CVCL_0030 HeLa WT Montreal Neurological Institute - CVCL_B6RU HeLa VAPB KO To screen the antibodies by western blot, WT and VAPB KO protein lysates were ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with all six VAPB antibodies in parallel ( Figure 1 ). Figure 1. VAPB antibody screening by western blot. Lysates of HeLa WT and VAPB KO were prepared, and 40 μg of protein were processed for western blot with the indicated VAPB antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Antibody dilution used: ab315013** at 1/1000, A5363 at 1/500, GTX131631 at 1/1000, 66191-1-Ig* at 1/1000, MA5-29639** at 1/1000, PA5-53023 at 1/1500 (0.2 μg/ml). Predicted band size: 27.2 kDa. **Recombinant antibody, *Monoclonal antibody. We then assessed the capability of the six antibodies to capture VAPB from HeLa protein extracts using immunoprecipitation techniques, followed by western blot analysis. For the immunoblotting step, a specific VAPB antibody identified previously (refer to Figure 1 ) was selected. Equal amounts of the starting material (SM) and the unbound fractions (UB), as well as the whole immunoprecipitate (IP) eluates were separated by SDS-PAGE ( Figure 2 ). Figure 2. VAPB antibody screening by immunoprecipitation. HeLa lysates were prepared, and immunoprecipitation was performed using 1 mg of lysate and 2.0 μg of the indicated VAPB antibodies pre-coupled to Dynabeads protein A or protein G. Samples were washed and processed for western blot with the indicated VAPB antibody. For western blot, ab315013** was used at 1/1000. The Ponceau stained transfers of each blot are shown. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate. **Recombinant antibody, *Monoclonal antibody. For immunofluorescence, the six antibodies were screened using a mosaic strategy. First, HeLa WT and VAPB KO cells were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the VAPB antibodies were evaluated. Both WT and KO lines imaged in the same field of view to reduce staining, imaging and image analysis bias ( Figure 3 ). Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested, and the images presented in Figure 3 are representative of this analysis. 7 Figure 3. VAPB antibody screening by immunofluorescence. HeLa WT and VAPB KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio on coverslips. Cells were stained with the indicated VAPB antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (WT), red (antibody staining) and far-red (KO) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed lines, respectively. When an antibody was recommended for immunofluorescence by the supplier, we tested it at the recommended dilution. The rest of the antibodies were tested at 1 and 2 μg/ml and the final concentration was selected based on the detection range of the microscope used and a quantitative analysis not shown here. Antibody dilution used: ab315013** at 1/250, A5363 at 1/1400, GTX131631 at 1/1000, 66191-1-Ig* at 1/800, MA5-29639** at 1/1000, PA5-53023 at 1/150. Bars = 10 μm. **Recombinant antibody, *Monoclonal antibody. In conclusion, we have screened six VAPB commercial antibodies by western blot, immunoprecipitation, and immunofluorescence by comparing the signal produced by the antibodies in human HeLa WT and VAPB KO cells. To assist users in interpreting antibody performance, Table 3 outlines various scenarios in which antibodies may perform in all three applications. 4 Several high-quality and renewable antibodies that successfully detect VAPB were identified in all applications. Researchers who wish to study VAPB in a different species are encouraged to select high-quality antibodies, based on the results of this study, and investigate the predicted species reactivity of the manufacturer before extending their research. The underlying data for this study can be found on Zenodo, an open-access repository for which YCharOS has its own collection of antibody characterization reports. Limitations Inherent limitations are associated with the antibody characterization platform used in this study. Firstly, the YCharOS project focuses on renewable (recombinant and monoclonal) antibodies and does not test all commercially available VAPB antibodies. YCharOS partners provide approximately 80% of all renewable antibodies, but some top-cited polyclonal antibodies may not be available through these partners. Secondly, the YCharOS effort employs a non-biased approach that is agnostic to the protein for which antibodies have been characterized. The aim is to provide objective data on antibody performance without preconceived notions about how antibodies should perform or the molecular weight that should be observed in western blot. As the authors are not experts in VAPB, only a brief overview of the protein’s function and its relevance in disease is provided. VAPB experts are invited to analyze and interpret observed banding patterns in western blots and subcellular localization in immunofluorescence. Thirdly, YCharOS experiments are not performed in replicates primarily due to the use of multiple antibodies targeting various epitopes. Once a specific antibody is identified, it validates the protein expression of the intended target in the selected cell line, confirms the lack of protein expression in the KO cell line and supports conclusions regarding the specificity of the other antibodies. All experiments are performed using master mixes, and meticulous attention is paid to sample preparation and experimental execution. In IF, the use of two different concentrations serves to evaluate antibody specificity and can aid in assessing assay reliability. In instances where antibodies yield no signal, a repeat experiment is conducted following titration. Additionally, our independent data is performed subsequently to the antibody manufacturers internal validation process, therefore making our characterization process a repeat. Lastly, as comprehensive and standardized procedures are respected, any conclusions remain confined to the experimental conditions and cell line used for this study. The use of a single cell type for evaluating antibody performance poses as a limitation, as factors such as target protein abundance significantly impact results. 7 Additionally, the use of cancer cell lines containing gene mutations poses a potential challenge, as these mutations may be within the epitope coding sequence or other regions of the gene responsible for the intended target. Such alterations can impact the binding affinity of antibodies. This represents an inherent limitation of any approach that employs cancer cell lines. Method The standardized protocols used to carry out this KO cell line-based antibody characterization platform was established and approved by a collaborative group of academics, industry researchers and antibody manufacturers. The detailed materials and step-by-step protocols used to characterize antibodies in western blot, immunoprecipitation and immunofluorescence are openly available on Protocol Exchange, a preprint server (DOI: 10.21203/rs.3.pex-2607/v1 ). 7 Brief descriptions of the experimental setup used to carry out this study can be found below. Cell lines and antibodies Cell lines used and primary antibodies tested in this study are listed in Tables 1 and 2 , respectively. To ensure that the cell lines and antibodies are cited properly and can be easily identified, we have included their corresponding Research Resource Identifiers, or RRID. 11 , 12 HeLa VAPB KO clone was generated at the Early Drug Discovery Unit (EDDU) using a guide RNA with the following sequence: UGAAGACUACAGCACCACGU. Table 2. Summary of the VAPB antibodies tested. Company Catalog number Lot number RRID (Antibody Registry) Clonality Clone ID Host Concentration (μg/μL) Vendors recommended applications Abcam ab315013 ** 1075876-3 AB_3086770 recombinant-mono EPR27026-50 rabbit 0.51 Wb, IP ABclonal A5363 12860201 AB_2766173 polyclonal - rabbit 1.41 Wb, IF GeneTex GTX131631 43103 AB_2886511 polyclonal rabbit 0.47 Wb, IP, IF Proteintech 66191-1-Ig * 10004068 AB_2881586 monoclonal 4F6A6 mouse 1.40 Wb, IF Thermo Fisher Scientific MA5-29639 ** WA3152374 AB_2785468 recombinant-mono 208 rabbit 1.00 Wb Thermo Fisher Scientific PA5-53023 YF3969135A AB_2649391 polyclonal - rabbit 0.30 Wb, IP, IF * Monoclonal antibody. ** Recombinant antibody. Table 3. Illustrations to assess antibody performance in all western blot, immunoprecipitation and immunofluorescence. Western blot Immunoprecipitation Immunofluorescence Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are (Thermo Fisher Scientific, cat. number 65-6120 and 62-6520). Peroxidase-conjugated VeriBlot for IP detection is from Abcam, cat. number ab131366. Alexa-555-conjugated goat anti-rabbit and anti-mouse secondary antibodies (Thermo Fisher Scientific, cat. number A21429 and A21424). Antibody screening by western blot HeLa WT and VAPB KO cells were collected in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Thermo Fisher Scientific, cat. number 89901) supplemented with 1× protease inhibitor cocktail mix (MilliporeSigma, cat. number P8340). Lysates were sonicated briefly and incubated 30 min on ice. Lysates were spun at ~110,000 × g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and western blot. BLUelf prestained protein ladder (GeneDireX, cat. number PM008-0500) was used. Western blots were performed with precast midi 4-20% Tris-Glycine polyacrylamide gels (Thermo Fisher Scientific, cat. number WXP42012BOX) ran with Tris/Glycine/SDS buffer (Bio-Rad, cat. number 1610772), loaded in Laemmli loading sample buffer (Thermo Fisher Scientific, cat. number AAJ61337AD) and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau S staining (Thermo Fisher Scientific, cat. number BP103-10) which is scanned to show together with individual western blot. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated O/N at 4°C with 5% milk in TBS with 0.1% Tween 20 (TBST) (Cell Signalling Technology, cat. number 9997). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 μg/ml in TBST with 5% milk for 1 hr at room temperature followed by three washes with TBST. Membranes were incubated with Pierce ECL (Thermo Fisher Scientific, cat. number 32106) prior to detection with the iBright™ CL1500 Imaging System (Thermo Fisher Scientific, cat. number A44240). Antibody screening by immunoprecipitation Antibody-bead conjugates were prepared by adding 2 μg of antibody to 500 μl of Pierce IP Lysis Buffer from Thermo Fisher Scientific (cat. number 87788) in a microcentrifuge tube, together with 30 μl of Dynabeads protein A- (for rabbit antibodies) or protein G- (for mouse antibodies) (Thermo Fisher Scientific, cat. number 10002D and 10004D, respectively). Tubes were rocked for ~1 h at 4°C followed by two washes to remove unbound antibodies. HeLa WT were collected in Pierce IP buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) supplemented with protease inhibitor. Lysates were rocked 30 min at 4°C and spun at 110,000 × g for 15 min at 4°C. 0.5 ml aliquots at 2 mg/ml of lysate were incubated with an antibody-bead conjugate for ~1 h at 4°C. The unbound fractions were collected, and beads were subsequently washed three times with 1.0 ml of IP buffer and processed for SDS-PAGE and western blot on a precast midi 4-20% Tris-Glycine polyacrylamide gels. VeriBlot for IP Detection Reagent:HRP was used as a secondary detection system at a concentration of 0.1 μg/ml. Antibody screening by immunofluorescence HeLa WT and VAPB KO cells were labelled with a green and a far-red fluorescence dye, respectively (Thermo Fisher Scientific, cat. number C2925 and C34565). The nuclei were labelled with DAPI (Thermo Fisher Scientific, cat. Number D3571) fluorescent stain. WT and KO cells were plated on 96-well plate with optically clear flat-bottom (Perkin Elmer, cat. number 6055300) as a mosaic and incubated for 24 hrs in a cell culture incubator at 37 o C, 5% CO 2 . Cells were fixed in 4% paraformaldehyde (PFA) (VWR, cat. number 100503-917) in phosphate buffered saline (PBS) (Wisent, cat. number 311-010-CL). Cells were permeabilized in PBS with 0.1% Triton X-100 (Thermo Fisher Scientific, cat. number BP151-500) for 10 min at room temperature and blocked with PBS with 5% BSA, 5% goat serum (Gibco, cat. number 16210-064) and 0.01% Triton X-100 for 30 min at room temperature. Cells were incubated with IF buffer (PBS, 5% BSA, 0.01% Triton X-100) containing the primary VAPB antibodies overnight at 4°C. Cells were then washed 3 × 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 μg/ml for 1 hr at room temperature with DAPI. Cells were washed 3 × 10 min with IF buffer and once with PBS. Images were acquired on an ImageXpress micro confocal high-content microscopy system (Molecular Devices), using a 20x NA 0.95 water immersion objective and scientific CMOS cameras, equipped with 395, 475, 555 and 635 nm solid state LED lights (lumencor Aura III light engine) and bandpass filters to excite DAPI, Cellmask Green, Alexa-555 and Cellmask Red, respectively. Images had pixel sizes of 0.68 × 0.68 microns, and a z-interval of 4 microns. For analysis and visualization, shading correction (shade only) was carried out for all images. Then, maximum intensity projections were generated using 3 z-slices. Segmentation was carried out separately on maximum intensity projections of Cellmask channels using CellPose 1.0, and masks were used to generate outlines and for intensity quantification. 13 Figures were assembled with Adobe Illustrator. Data availability Underlying data Zenodo: Antibody Characterization Report for VAPB, https://doi.org/10.5281/zenodo.13891570 . 14 Zenodo: Dataset for the VAPB antibody screening study, https://doi.org/10.5281/zenodo.14503380 . 15 Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Acknowledgment We would like to thank the NeuroSGC/YCharOS/EDDU collaborative group for their important contribution to the creation of an open scientific ecosystem of antibody manufacturers and KO cell line suppliers, for the development of community-agreed protocols, and for their shared ideas, resources, and collaboration. Members of the group can be found below. We would also like to thank the Advanced BioImaging Facility (ABIF) consortium for their image analysis pipeline development and conduction (RRID:SCR_017697). Members of each group can be found below. NeuroSGC/YCharOS/EDDU collaborative group: Thomas M. Durcan, Aled M. Edwards, Peter S. McPherson, Chetan Raina and Wolfgang Reintsch. ABIF consortium: Claire M. Brown and Joel Ryan. Thank you to the Structural Genomics Consortium, a registered charity (no. 1097737), for your support on this project. The Structural Genomics Consortium receives funding from Bayer AG, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute (grant no. OGI-196), the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no. 875510), Janssen, Merck KGaA (also known as EMD in Canada and the United States), Pfizer and Takeda. References 1. Nishimura Y, Hayashi M, Inada H, et al. : Molecular cloning and characterization of mammalian homologues of vesicle-associated membrane protein-associated (VAMP-associated) proteins. Biochem. Biophys. Res. Commun. 1999; 254 (1): 21–26. PubMed Abstract | Publisher Full Text 2. Teuling E, Ahmed S, Haasdijk E, et al. : Motor neuron disease-associated mutant vesicle-associated membrane protein-associated protein (VAP) B recruits wild-type VAPs into endoplasmic reticulum-derived tubular aggregates. J. Neurosci. 2007; 27 (36): 9801–9815. PubMed Abstract | Publisher Full Text | Free Full Text 3. Nishimura AL, Mitne-Neto M, Silva HC, et al. : A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. Am. J. Hum. Genet. 2004; 75 (5): 822–831. PubMed Abstract | Publisher Full Text | Free Full Text 4. Ayoubi R, Ryan J, Biddle MS, et al. : Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications. elife. 2023; 12 : 12. Publisher Full Text 5. Carter AJ, Kraemer O, Zwick M, et al. : Target 2035: probing the human proteome. Drug Discov. Today. 2019; 24 (11): 2111–2115. PubMed Abstract | Publisher Full Text 6. Licciardello MP, Workman P: The era of high-quality chemical probes. RSC Med. Chem. 2022; 13 (12): 1446–1459. PubMed Abstract | Publisher Full Text | Free Full Text 7. Ayoubi R, Ryan J, Bolivar SG, et al. : A consensus platform for antibody characterization (Version 1). Protocol. Exchange. 2024. 8. Biddle MS, Virk HS: YCharOS open antibody characterisation data: Lessons learned and progress made. F1000Res. 2023; 12 (12): 1344. Publisher Full Text 9. Laflamme C, McKeever PM, Kumar R, et al. : Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. elife. 2019; 8 : 8. Publisher Full Text 10. Alshafie W, Fotouhi M, Shlaifer I, et al. : Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence. F1000Res. 2022; 11 : 977. Publisher Full Text 11. Bandrowski A, Pairish M, Eckmann P, et al. : The Antibody Registry: ten years of registering antibodies. Nucleic Acids Res. 2023; 51 (D1): D358–D367. PubMed Abstract | Publisher Full Text | Free Full Text 12. Bairoch A: The Cellosaurus, a Cell-Line Knowledge Resource. J. Biomol. Tech. 2018; 29 (2): 25–38. PubMed Abstract | Publisher Full Text | Free Full Text 13. Stringer C, Wang T, Michaelos M, et al. : Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods. 2021; 18 (1): 100–106. PubMed Abstract | Publisher Full Text 14. Gonzalez Bolivar S, Ayoubi R, Alende C, et al. : A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence.2024. Publisher Full Text 15. Laflamme C: Dataset for the VAPB antibody screening study. [Dataset]. Zenodo. 2024. Publisher Full Text Comments on this article Comments (0) Version 1 VERSION 1 PUBLISHED 24 Dec 2024 ADD YOUR COMMENT Comment Author details Author details 1 Montreal Neurological Institute-Hospital, Montreal, Québec, Canada Sara González Bolívar Roles: Investigation Riham Ayoubi Roles: Investigation, Supervision, Writing – Original Draft Preparation Charles Alende Roles: Investigation Maryam Fothouhi Roles: Investigation Irina Shlaifer Roles: Investigation Peter S. McPherson Roles: Funding Acquisition Carl Laflamme Roles: Conceptualization, Funding Acquisition, Writing – Review & Editing Competing interests For this project, the authors developed partnerships with high-quality antibody manufacturers and KO cell line providers. The partners provide antibodies and KO cell lines to the McPherson laboratory at no cost. These partners include: - Abcam-ABCD antibodies- ABclonal- Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific. Grant information This work was supported by a grant from the Motor Neurone Disease Association (UK), The ALS Association (USA) and ALS Canada, by a Canadian Institutes of Health Research Foundation Grant (FDN154305) and by the Government of Canada through Genome Canada and Ontario Genomics (OGI-210). RA is supported by a Mitacs fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (1) version 1 Published: 24 Dec 2024, 13:1559 https://doi.org/10.12688/f1000research.160226.1 Copyright © 2024 González Bolívar S et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article González Bolívar S, Ayoubi R, Alende C et al. A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 3 approved, 1 approved with reservations] . F1000Research 2024, 13 :1559 ( https://doi.org/10.12688/f1000research.160226.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 24 Dec 2024 Views 0 Cite How to cite this report: Shao J. Reviewer Report For: A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 3 approved, 1 approved with reservations] . F1000Research 2024, 13 :1559 ( https://doi.org/10.5256/f1000research.176085.r359974 ) The direct URL for this report is: https://f1000research.com/articles/13-1559/v1#referee-response-359974 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 05 Feb 2025 Jieya Shao , Washington University in St Louis, St. Louis, Missouri, USA Approved VIEWS 0 https://doi.org/10.5256/f1000research.176085.r359974 In this article the authors systematically compared the performance and specificity of six commercial anti-VAPB antibodies using three different experimental approaches (Western blot, immunoprecipitation, and immunofluorescence staining). This study is part of a large collaborative initiative to characterize commercial antibodies ... Continue reading READ ALL In this article the authors systematically compared the performance and specificity of six commercial anti-VAPB antibodies using three different experimental approaches (Western blot, immunoprecipitation, and immunofluorescence staining). This study is part of a large collaborative initiative to characterize commercial antibodies for different human proteins and offer unbiased guidance to researchers who can select the best antibodies for their specific experimental objectives based on their own interpretations. As a Data Note, this paper successfully achieved its goal with rigorously collected high-quality data, and the results are self-explanatory and useful for the research community. No issues were found except a small grammatical error on page 3 under Results and discussion. The authors wrote "WT and VAPB KO protein lysates were ran on SDS PAGE". The correct word is "run". Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: DNA replication, cancer biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Shao J. Reviewer Report For: A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 3 approved, 1 approved with reservations] . F1000Research 2024, 13 :1559 ( https://doi.org/10.5256/f1000research.176085.r359974 ) The direct URL for this report is: https://f1000research.com/articles/13-1559/v1#referee-response-359974 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Bowman K. Reviewer Report For: A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 3 approved, 1 approved with reservations] . F1000Research 2024, 13 :1559 ( https://doi.org/10.5256/f1000research.176085.r359968 ) The direct URL for this report is: https://f1000research.com/articles/13-1559/v1#referee-response-359968 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 05 Feb 2025 Karen Bowman , University of Leicester, Leicester, England, UK Approved VIEWS 0 https://doi.org/10.5256/f1000research.176085.r359968 The study involved characterisation of six commercial antibodies, from five different suppliers, for the vesicle-associated membrane protein-associated protein B/C (VAPB) for use in western blot, immunoprecipitation, and immunofluorescence. The study formed part of the wider YCharOS collaboration to characterise commercial ... Continue reading READ ALL The study involved characterisation of six commercial antibodies, from five different suppliers, for the vesicle-associated membrane protein-associated protein B/C (VAPB) for use in western blot, immunoprecipitation, and immunofluorescence. The study formed part of the wider YCharOS collaboration to characterise commercial antibodies for human proteins using standardised protocols with the intention of improving antibody reproducibility issues. As a Data Note, it allows the direct comparison of the performance of commercially available antibodies to VAPB protein to aid scientists in choosing the most appropriate antibody for their studies, for example, in the biochemical and cellular assessment of VAPB properties and function. This comparison would be of particular use in the field of neurodegenerative diseases, especially in the study of amyotrophic lateral sclerosis (ALS). A committee of industry and academic representatives have endorsed the platform used, and the protocols used are consistent with those in general use. The platform consisted of identification of a human cell line suitable for antibody characterisation studies i.e., with adequate levels of the VAPB protein to generate a measurable signal. They found that the commercially available HeLa WT cell line expressed VAPB transcript at a level above the average range of cancer cells analysed, and therefore, was a logical choice for their study. The matched isogenic knockout control cell line, HeLa VAPB KO was used as an appropriate negative control. The final step was a series of antibody characterisation procedures, limited to the most common research uses of antibodies. The interpretation of the results is left to the reader, with the study providing unbiased guidance. However, it gave some indication of the optimum dilutions of commercial antibodies. Limitations of the study are clearly stated. The summary Table 3 with illustrations to assess antibody performance in western blot, immunoprecipitation and immunofluorescence, is very helpful. I think this study could have been improved by increasing the number of commercially available antibodies studied. There are at least a further seven commercial VAPB antibodies available. However, the study is a useful starting point when choosing the most appropriate antibody for study of the VAPB protein. Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: cell biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Bowman K. Reviewer Report For: A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 3 approved, 1 approved with reservations] . F1000Research 2024, 13 :1559 ( https://doi.org/10.5256/f1000research.176085.r359968 ) The direct URL for this report is: https://f1000research.com/articles/13-1559/v1#referee-response-359968 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Tao Sc. Reviewer Report For: A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 3 approved, 1 approved with reservations] . F1000Research 2024, 13 :1559 ( https://doi.org/10.5256/f1000research.176085.r359970 ) The direct URL for this report is: https://f1000research.com/articles/13-1559/v1#referee-response-359970 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 24 Jan 2025 Sheng-ce Tao , Shanghai Jiao Tong University, Shanghai, China Approved VIEWS 0 https://doi.org/10.5256/f1000research.176085.r359970 Because of the importance of VAPB, high quality antibody is a needed. This study evaluates six commercial antibodies against VAPB for western blot, immunoprecipitation, and immunofluorescence using standardized protocols in knockout and isogenic control cell lines. The research aims to ... Continue reading READ ALL Because of the importance of VAPB, high quality antibody is a needed. This study evaluates six commercial antibodies against VAPB for western blot, immunoprecipitation, and immunofluorescence using standardized protocols in knockout and isogenic control cell lines. The research aims to address antibody reproducibility and provide open data to help researchers select the best antibodies for their studies. 1. What are the criteria for selecting the six commercial antibodies? Please clarify. 2. This study only tested one cell line (HeLa). It is not necessary that the conclusion from one cell generally also works among other cell lines. 3. Since one of the major application of these validated antibodies is WB, it is highly possible that these antibodies recognize linear epitopes on VAPB. In this case, mapping the epitopes (for example, using AbMap technology), and then judge the specificity by epitope searching across the whole proteome will be a more general approach. Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Antibody technology, proteomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Tao Sc. Reviewer Report For: A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 3 approved, 1 approved with reservations] . F1000Research 2024, 13 :1559 ( https://doi.org/10.5256/f1000research.176085.r359970 ) The direct URL for this report is: https://f1000research.com/articles/13-1559/v1#referee-response-359970 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 31 Jan 2025 Carl Laflamme , Montreal Neurological Institute-Hospital, Montreal, Canada 31 Jan 2025 Author Response Dear Dr. Sheng-ce Tao, Thank you for your review and feedback on our manuscript. In response to your points: The YCharOS initiative collaborates with 12 antibody ... Continue reading Dear Dr. Sheng-ce Tao, Thank you for your review and feedback on our manuscript. In response to your points: The YCharOS initiative collaborates with 12 antibody manufacturers, representing approximately 80% of all renewable antibodies commercially available. These partners have selected and provided their VAPB antibodies with us. We focus on renewable antibodies, as the YCharOS project does not undertake antibody characterization studies for proteins covered solely by polyclonal antibodies. The YCharOS workflow was recently featured and detailed in a News and Views article ( https://doi.org/10.1038/s41596-024-01108-6 ). We acknowledge the reviewer's concern that a limitation of this study is the screening of antibodies in a single cell line. Our study serves as an antibody selection guide, but it remains essential for researchers to validate antibody specificity and performance within their own experimental context. As stated in the limitations section: “ Lastly, as comprehensive and standardized procedures are respected, any conclusions remain confined to the experimental conditions and cell line used for this study. The use of a single cell type for evaluating antibody performance poses as a limitation, as factors such as target protein abundance significantly impact results (8). Additionally, the use of cancer cell lines containing gene mutations poses a potential challenge, as these mutations may be within the epitope coding sequence or other regions of the gene responsible for the intended target. Such alterations can impact the binding affinity of antibodies. This represents an inherent limitation of any approach that employs cancer cell lines ”. Mapping the epitopes of the tested antibodies falls outside the scope of the YCharOS initiative. Sincerely Carl Laflamme Dear Dr. Sheng-ce Tao, Thank you for your review and feedback on our manuscript. In response to your points: The YCharOS initiative collaborates with 12 antibody manufacturers, representing approximately 80% of all renewable antibodies commercially available. These partners have selected and provided their VAPB antibodies with us. We focus on renewable antibodies, as the YCharOS project does not undertake antibody characterization studies for proteins covered solely by polyclonal antibodies. The YCharOS workflow was recently featured and detailed in a News and Views article ( https://doi.org/10.1038/s41596-024-01108-6 ). We acknowledge the reviewer's concern that a limitation of this study is the screening of antibodies in a single cell line. Our study serves as an antibody selection guide, but it remains essential for researchers to validate antibody specificity and performance within their own experimental context. As stated in the limitations section: “ Lastly, as comprehensive and standardized procedures are respected, any conclusions remain confined to the experimental conditions and cell line used for this study. The use of a single cell type for evaluating antibody performance poses as a limitation, as factors such as target protein abundance significantly impact results (8). Additionally, the use of cancer cell lines containing gene mutations poses a potential challenge, as these mutations may be within the epitope coding sequence or other regions of the gene responsible for the intended target. Such alterations can impact the binding affinity of antibodies. This represents an inherent limitation of any approach that employs cancer cell lines ”. Mapping the epitopes of the tested antibodies falls outside the scope of the YCharOS initiative. Sincerely Carl Laflamme Competing Interests: none Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 31 Jan 2025 Carl Laflamme , Montreal Neurological Institute-Hospital, Montreal, Canada 31 Jan 2025 Author Response Dear Dr. Sheng-ce Tao, Thank you for your review and feedback on our manuscript. In response to your points: The YCharOS initiative collaborates with 12 antibody ... Continue reading Dear Dr. Sheng-ce Tao, Thank you for your review and feedback on our manuscript. In response to your points: The YCharOS initiative collaborates with 12 antibody manufacturers, representing approximately 80% of all renewable antibodies commercially available. These partners have selected and provided their VAPB antibodies with us. We focus on renewable antibodies, as the YCharOS project does not undertake antibody characterization studies for proteins covered solely by polyclonal antibodies. The YCharOS workflow was recently featured and detailed in a News and Views article ( https://doi.org/10.1038/s41596-024-01108-6 ). We acknowledge the reviewer's concern that a limitation of this study is the screening of antibodies in a single cell line. Our study serves as an antibody selection guide, but it remains essential for researchers to validate antibody specificity and performance within their own experimental context. As stated in the limitations section: “ Lastly, as comprehensive and standardized procedures are respected, any conclusions remain confined to the experimental conditions and cell line used for this study. The use of a single cell type for evaluating antibody performance poses as a limitation, as factors such as target protein abundance significantly impact results (8). Additionally, the use of cancer cell lines containing gene mutations poses a potential challenge, as these mutations may be within the epitope coding sequence or other regions of the gene responsible for the intended target. Such alterations can impact the binding affinity of antibodies. This represents an inherent limitation of any approach that employs cancer cell lines ”. Mapping the epitopes of the tested antibodies falls outside the scope of the YCharOS initiative. Sincerely Carl Laflamme Dear Dr. Sheng-ce Tao, Thank you for your review and feedback on our manuscript. In response to your points: The YCharOS initiative collaborates with 12 antibody manufacturers, representing approximately 80% of all renewable antibodies commercially available. These partners have selected and provided their VAPB antibodies with us. We focus on renewable antibodies, as the YCharOS project does not undertake antibody characterization studies for proteins covered solely by polyclonal antibodies. The YCharOS workflow was recently featured and detailed in a News and Views article ( https://doi.org/10.1038/s41596-024-01108-6 ). We acknowledge the reviewer's concern that a limitation of this study is the screening of antibodies in a single cell line. Our study serves as an antibody selection guide, but it remains essential for researchers to validate antibody specificity and performance within their own experimental context. As stated in the limitations section: “ Lastly, as comprehensive and standardized procedures are respected, any conclusions remain confined to the experimental conditions and cell line used for this study. The use of a single cell type for evaluating antibody performance poses as a limitation, as factors such as target protein abundance significantly impact results (8). Additionally, the use of cancer cell lines containing gene mutations poses a potential challenge, as these mutations may be within the epitope coding sequence or other regions of the gene responsible for the intended target. Such alterations can impact the binding affinity of antibodies. This represents an inherent limitation of any approach that employs cancer cell lines ”. Mapping the epitopes of the tested antibodies falls outside the scope of the YCharOS initiative. Sincerely Carl Laflamme Competing Interests: none Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Miklós MG. Reviewer Report For: A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 3 approved, 1 approved with reservations] . F1000Research 2024, 13 :1559 ( https://doi.org/10.5256/f1000research.176085.r357897 ) The direct URL for this report is: https://f1000research.com/articles/13-1559/v1#referee-response-357897 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 15 Jan 2025 Mórotz Gábor Miklós , Semmelweis University, Budapest, Budapest, Hungary Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.176085.r357897 In this paper Gonzales Bolivar and colleagues tested six commercially available anti-VAPB antibodies as part of the YCharOS initiative. The experimental results are good quality and clearly presented. These data allow fellow researchers to directly compare the performance of the ... Continue reading READ ALL In this paper Gonzales Bolivar and colleagues tested six commercially available anti-VAPB antibodies as part of the YCharOS initiative. The experimental results are good quality and clearly presented. These data allow fellow researchers to directly compare the performance of the selected antibodies side-by-side. I have only a few comments. 1. In the introduction section, please use more appropriate references for the cellular function of VAPB (e.g. Dudás EF, et al., 2021 [Ref 1]). Additionally, the major cellular functions regulated by VAPB tethering are lipid metabolism and calcium homeostasis. 2. It is not clear for me what were the exact antibody selection criteria for the study. For example, I can see monoclonal, recombinant and polyclonal antibodies on the list so clearly this was not a selection criterion although in the Limitations section is written that “project focuses on renewable (recombinant and monoclonal) antibodies”. It would be nice to add a flow-chart summarising how many antibodies are on the market and based on what criteria were these six antibodies selected for the work. In this way, the reader can decide how comprehensive this study is. 3. VAPB is an integral ER membrane protein. Therefore, for the immunofluorescent experiments it would be very informative to use an ER staining instead of a general cell staining to analyse whether the tested antibodies recognise the protein in the ER. 4. In the Results and discussion section first paragraph please correct “HeLa expresses the VAPB” to HeLa cells express the VAPB. 5. In the Methods section please correct the “peroxidase-conjugated antibodies” to horseradish peroxidase-conjugated antibodies. I also suggest using h instead of hr or hrs for the abbreviation of hour as it is more widely used in the scientific literature. Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes References 1. Dudás EF, Huynen MA, Lesk AM, Pastore A: Invisible leashes: The tethering VAPs from infectious diseases to neurodegeneration. J Biol Chem . 2021; 296 : 100421 PubMed Abstract | Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: neurodegeneration, VAPB I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Miklós MG. Reviewer Report For: A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 3 approved, 1 approved with reservations] . F1000Research 2024, 13 :1559 ( https://doi.org/10.5256/f1000research.176085.r357897 ) The direct URL for this report is: https://f1000research.com/articles/13-1559/v1#referee-response-357897 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Comments on this article Comments (0) Version 1 VERSION 1 PUBLISHED 24 Dec 2024 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 4 Version 1 24 Dec 24 read read read read Mórotz Gábor Miklós , Semmelweis University, Budapest, Hungary Sheng-ce Tao , Shanghai Jiao Tong University, Shanghai, China Karen Bowman , University of Leicester, Leicester, UK Jieya Shao , Washington University in St Louis, St. Louis, USA Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Shao J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 05 Feb 2025 | for Version 1 Jieya Shao , Washington University in St Louis, St. Louis, Missouri, USA 0 Views copyright © 2025 Shao J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions In this article the authors systematically compared the performance and specificity of six commercial anti-VAPB antibodies using three different experimental approaches (Western blot, immunoprecipitation, and immunofluorescence staining). This study is part of a large collaborative initiative to characterize commercial antibodies for different human proteins and offer unbiased guidance to researchers who can select the best antibodies for their specific experimental objectives based on their own interpretations. As a Data Note, this paper successfully achieved its goal with rigorously collected high-quality data, and the results are self-explanatory and useful for the research community. No issues were found except a small grammatical error on page 3 under Results and discussion. The authors wrote "WT and VAPB KO protein lysates were ran on SDS PAGE". The correct word is "run". Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise DNA replication, cancer biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Shao J. Peer Review Report For: A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 3 approved, 1 approved with reservations] . F1000Research 2024, 13 :1559 ( https://doi.org/10.5256/f1000research.176085.r359974) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1559/v1#referee-response-359974 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Bowman K. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 05 Feb 2025 | for Version 1 Karen Bowman , University of Leicester, Leicester, England, UK 0 Views copyright © 2025 Bowman K. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The study involved characterisation of six commercial antibodies, from five different suppliers, for the vesicle-associated membrane protein-associated protein B/C (VAPB) for use in western blot, immunoprecipitation, and immunofluorescence. The study formed part of the wider YCharOS collaboration to characterise commercial antibodies for human proteins using standardised protocols with the intention of improving antibody reproducibility issues. As a Data Note, it allows the direct comparison of the performance of commercially available antibodies to VAPB protein to aid scientists in choosing the most appropriate antibody for their studies, for example, in the biochemical and cellular assessment of VAPB properties and function. This comparison would be of particular use in the field of neurodegenerative diseases, especially in the study of amyotrophic lateral sclerosis (ALS). A committee of industry and academic representatives have endorsed the platform used, and the protocols used are consistent with those in general use. The platform consisted of identification of a human cell line suitable for antibody characterisation studies i.e., with adequate levels of the VAPB protein to generate a measurable signal. They found that the commercially available HeLa WT cell line expressed VAPB transcript at a level above the average range of cancer cells analysed, and therefore, was a logical choice for their study. The matched isogenic knockout control cell line, HeLa VAPB KO was used as an appropriate negative control. The final step was a series of antibody characterisation procedures, limited to the most common research uses of antibodies. The interpretation of the results is left to the reader, with the study providing unbiased guidance. However, it gave some indication of the optimum dilutions of commercial antibodies. Limitations of the study are clearly stated. The summary Table 3 with illustrations to assess antibody performance in western blot, immunoprecipitation and immunofluorescence, is very helpful. I think this study could have been improved by increasing the number of commercially available antibodies studied. There are at least a further seven commercial VAPB antibodies available. However, the study is a useful starting point when choosing the most appropriate antibody for study of the VAPB protein. Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise cell biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Bowman K. Peer Review Report For: A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 3 approved, 1 approved with reservations] . F1000Research 2024, 13 :1559 ( https://doi.org/10.5256/f1000research.176085.r359968) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1559/v1#referee-response-359968 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Tao S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 24 Jan 2025 | for Version 1 Sheng-ce Tao , Shanghai Jiao Tong University, Shanghai, China 0 Views copyright © 2025 Tao S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Because of the importance of VAPB, high quality antibody is a needed. This study evaluates six commercial antibodies against VAPB for western blot, immunoprecipitation, and immunofluorescence using standardized protocols in knockout and isogenic control cell lines. The research aims to address antibody reproducibility and provide open data to help researchers select the best antibodies for their studies. 1. What are the criteria for selecting the six commercial antibodies? Please clarify. 2. This study only tested one cell line (HeLa). It is not necessary that the conclusion from one cell generally also works among other cell lines. 3. Since one of the major application of these validated antibodies is WB, it is highly possible that these antibodies recognize linear epitopes on VAPB. In this case, mapping the epitopes (for example, using AbMap technology), and then judge the specificity by epitope searching across the whole proteome will be a more general approach. Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Antibody technology, proteomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (1) Author Response 31 Jan 2025 Carl Laflamme, Montreal Neurological Institute-Hospital, Montreal, Canada Dear Dr. Sheng-ce Tao, Thank you for your review and feedback on our manuscript. In response to your points: The YCharOS initiative collaborates with 12 antibody manufacturers, representing approximately 80% of all renewable antibodies commercially available. These partners have selected and provided their VAPB antibodies with us. We focus on renewable antibodies, as the YCharOS project does not undertake antibody characterization studies for proteins covered solely by polyclonal antibodies. The YCharOS workflow was recently featured and detailed in a News and Views article ( https://doi.org/10.1038/s41596-024-01108-6 ). We acknowledge the reviewer's concern that a limitation of this study is the screening of antibodies in a single cell line. Our study serves as an antibody selection guide, but it remains essential for researchers to validate antibody specificity and performance within their own experimental context. As stated in the limitations section: “ Lastly, as comprehensive and standardized procedures are respected, any conclusions remain confined to the experimental conditions and cell line used for this study. The use of a single cell type for evaluating antibody performance poses as a limitation, as factors such as target protein abundance significantly impact results (8). Additionally, the use of cancer cell lines containing gene mutations poses a potential challenge, as these mutations may be within the epitope coding sequence or other regions of the gene responsible for the intended target. Such alterations can impact the binding affinity of antibodies. This represents an inherent limitation of any approach that employs cancer cell lines ”. Mapping the epitopes of the tested antibodies falls outside the scope of the YCharOS initiative. Sincerely Carl Laflamme View more View less Competing Interests none reply Respond Report a concern Tao Sc. Peer Review Report For: A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 3 approved, 1 approved with reservations] . F1000Research 2024, 13 :1559 ( https://doi.org/10.5256/f1000research.176085.r359970) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1559/v1#referee-response-359970 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Miklós M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 15 Jan 2025 | for Version 1 Mórotz Gábor Miklós , Semmelweis University, Budapest, Budapest, Hungary 0 Views copyright © 2025 Miklós M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions In this paper Gonzales Bolivar and colleagues tested six commercially available anti-VAPB antibodies as part of the YCharOS initiative. The experimental results are good quality and clearly presented. These data allow fellow researchers to directly compare the performance of the selected antibodies side-by-side. I have only a few comments. 1. In the introduction section, please use more appropriate references for the cellular function of VAPB (e.g. Dudás EF, et al., 2021 [Ref 1]). Additionally, the major cellular functions regulated by VAPB tethering are lipid metabolism and calcium homeostasis. 2. It is not clear for me what were the exact antibody selection criteria for the study. For example, I can see monoclonal, recombinant and polyclonal antibodies on the list so clearly this was not a selection criterion although in the Limitations section is written that “project focuses on renewable (recombinant and monoclonal) antibodies”. It would be nice to add a flow-chart summarising how many antibodies are on the market and based on what criteria were these six antibodies selected for the work. In this way, the reader can decide how comprehensive this study is. 3. VAPB is an integral ER membrane protein. Therefore, for the immunofluorescent experiments it would be very informative to use an ER staining instead of a general cell staining to analyse whether the tested antibodies recognise the protein in the ER. 4. In the Results and discussion section first paragraph please correct “HeLa expresses the VAPB” to HeLa cells express the VAPB. 5. In the Methods section please correct the “peroxidase-conjugated antibodies” to horseradish peroxidase-conjugated antibodies. I also suggest using h instead of hr or hrs for the abbreviation of hour as it is more widely used in the scientific literature. Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes References 1. Dudás EF, Huynen MA, Lesk AM, Pastore A: Invisible leashes: The tethering VAPs from infectious diseases to neurodegeneration. J Biol Chem . 2021; 296 : 100421 PubMed Abstract | Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise neurodegeneration, VAPB I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Miklós MG. Peer Review Report For: A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 3 approved, 1 approved with reservations] . F1000Research 2024, 13 :1559 ( https://doi.org/10.5256/f1000research.176085.r357897) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1559/v1#referee-response-357897 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. 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