A guide to selecting high-performing antibodies for Heterogeneous nuclear ribonucleoproteins A2/B1(hnRNP A2/B1) (UniProt ID: P22626) for use in western blot, immunoprecipitation, and immunofluorescence

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McPherson" }, { "@type": "Person", "name": "Carl Laflamme" } ], "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": "Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) is a key RNA-binding protein involved in alternative splicing, mRNA transport, and local translation processes essential for neuronal development and synaptic plasticity. Here we have characterized eight hnRNP A2/B1 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 the 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/14-1158", "name": "A guide to selecting high-performing antibodies for Heterogeneous..." } } ] } Home Browse A guide to selecting high-performing antibodies for Heterogeneous... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Moleón VR, Ayoubi R, Alende C et al. A guide to selecting high-performing antibodies for Heterogeneous nuclear ribonucleoproteins A2/B1(hnRNP A2/B1) (UniProt ID: P22626) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 2 approved] . F1000Research 2025, 14 :1158 ( https://doi.org/10.12688/f1000research.170886.1 ) 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 Heterogeneous nuclear ribonucleoproteins A2/B1(hnRNP A2/B1) (UniProt ID: P22626) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 2 approved] Vera Ruíz Moleón https://orcid.org/0000-0003-3728-3158 1 , Riham Ayoubi 1 , Charles Alende https://orcid.org/0009-0005-4611-6134 1 , [...] Maryam Fothouhi 1 , Joel Ryan 2 , Sara González Bolívar https://orcid.org/0000-0002-4299-8281 1 , Donovan Worrall https://orcid.org/0000-0001-5676-5473 1 , Thomas M Durcan 1 , Claire M Brown 2 , Vincent Francis https://orcid.org/0009-0000-7535-8718 1 , Peter S. McPherson 1 , Carl Laflamme https://orcid.org/0000-0001-5906-025X 1 Vera Ruíz Moleón https://orcid.org/0000-0003-3728-3158 1 , Riham Ayoubi 1 , [...] Charles Alende https://orcid.org/0009-0005-4611-6134 1 , Maryam Fothouhi 1 , Joel Ryan 2 , Sara González Bolívar https://orcid.org/0000-0002-4299-8281 1 , Donovan Worrall https://orcid.org/0000-0001-5676-5473 1 , Thomas M Durcan 1 , Claire M Brown 2 , Vincent Francis https://orcid.org/0009-0000-7535-8718 1 , Peter S. McPherson 1 , Carl Laflamme https://orcid.org/0000-0001-5906-025X 1 PUBLISHED 23 Oct 2025 Author details Author details 1 Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital,Structural Genomics Consortium, ABIF consortium, Montreal, Québec, Canada 2 McGill University, Montreal, Québec, Canada Vera Ruíz Moleón Roles: Investigation Riham Ayoubi Roles: Investigation, Supervision, Writing – Original Draft Preparation Charles Alende Roles: Investigation Maryam Fothouhi Roles: Investigation Joel Ryan Roles: Investigation Sara González Bolívar Roles: Investigation Donovan Worrall Roles: Investigation Thomas M Durcan Roles: Investigation Claire M Brown Roles: Investigation Vincent Francis Roles: Writing – Original Draft Preparation, Writing – Review & Editing Peter S. McPherson Roles: Funding Acquisition, Supervision 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 Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) is a key RNA-binding protein involved in alternative splicing, mRNA transport, and local translation processes essential for neuronal development and synaptic plasticity. Here we have characterized eight hnRNP A2/B1 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 the 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 P22626, HNRNPA2B1, Heterogeneous nuclear ribonucleoproteins A2/B1, 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 leading antibody manufacturers and KO cell line providers. The partners provide antibodies and KO cell lines to this project at no cost. These partners include: Abcam, ABCD antibodies, ABclonal, Addgene, Aviva Systems Biology, BioTechne, Cell Signaling Technology, Developmental Studies Hybridoma Bank, GeneTex, Horizon Discovery, Institute for Protein Innovation, MilliporeSigma, Proteintech, Synaptic Systems, Thermo Fisher Scientific. Grant information: This work was supported in part by a grant from the ALS-Reproducible Antibody Platform (ALS-RAP). ALS-RAP was created as a public-private partnership by three leading ALS charities: the ALS Association (USA), the Motor Neurone Disease Association (UK), and the ALS Society of Canada. This work was also supported by the Government of Canada through Genome Canada, Genome Quebec, and Ontario Genomics (grant no. OGI-210). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 Moleón VR 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: Moleón VR, Ayoubi R, Alende C et al. A guide to selecting high-performing antibodies for Heterogeneous nuclear ribonucleoproteins A2/B1(hnRNP A2/B1) (UniProt ID: P22626) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 2 approved] . F1000Research 2025, 14 :1158 ( https://doi.org/10.12688/f1000research.170886.1 ) First published: 23 Oct 2025, 14 :1158 ( https://doi.org/10.12688/f1000research.170886.1 ) Latest published: 23 Oct 2025, 14 :1158 ( https://doi.org/10.12688/f1000research.170886.1 ) Introduction hnRNP A2/B1 is an essential RNA-binding protein that regulates mRNA splicing, stability, and axonal transport in neurons. 1 Highly enriched in the central nervous system, hnRNP A2/B1 regulates the transport of mRNAs to dendrites and axons, where localized translation supports rapid, activity-dependent responses. 2 Pathogenic mutations and misregulation of hnRNP A2/B1 have been linked to neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and multiple system atrophy. Notably, mutations in the HNRNPA2B1 gene can lead to prion-like aggregation and cytoplasmic mislocalization, disrupting normal RNA metabolism and protein homeostasis. 3 hnRNP A2/B1 also interacts with stress granules and shares functional similarities with other ALS-linked proteins such as TDP-43 and FUS. 4 Its dual role in maintaining RNA dynamics and 5 vulnerability and suggests potential therapeutic avenues targeting RNA-protein homeostasis. 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. 5 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 renewable antibodies against the corresponding protein. 5 Here we characterized eight commercial hnRNP A2/B1 antibodies, selected and donated by participant antibody manufacturers, for use in western blot, immunoprecipitation, and immunofluorescence (also referred to as immunocytochemistry), enabling biochemical and cellular assessment of hnRNP A2/B1 properties and function. The platform for antibody characterization used to carry out this study was endorsed by a committee of industry academic representatives. The standardized consensus antibody characterization protocols are openly available on Protocol Exchange. 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 6 and in Table 4 of this data note. 5 Table 1. Summary of the cell lines used. Institution Catalog number RRID (Cellosaurus) Cell line Genotype Horizon Discovery C631 CVCL_Y019 HAP1 WT Horizon Discovery HZGHC007378c010 CVCL_C9EQ HAP1 HNRNPA2B1 KO Table 2. Summary of the hnRNP A2/B1 antibodies tested. Company Catalog number Lot number RRID (Antibody Registry) Clonality Clone ID Host Concentration (μg/μl) Vendors recommended applications Bio-Techne NB120-6102 * B1 AB_790226 monoclonal DP3B3 mouse 1 Wb, IP, IF Bio-Techne NBP2-80777 * B1 AB_2943694 monoclonal DP3B3 mouse 1 Wb, IP, IF Cell Signaling Technology 9304 * 2 AB_10694208 monoclonal 2A2 mouse n/a Wb Genetex GTX114475 40618 AB_11165592 polyclonal - rabbit 0.75 Wb, IF Genetex GTX127928 44573 AB_2616069 polyclonal - rabbit 1.09 Wb, IP, IF Genetex GTX637305 ** 44837 AB_2943695 recombinant-mono HL1706 rabbit 1 Wb, IF Proteintech 67445-1-Ig * 10027542 AB_2882679 monoclonal 3H6F7 mouse 1.8 Wb, IF Thermo Fisher scientific PA5-81963 YE3914206A AB_2789124 polyclonal - rabbit 0.1 Wb ** = Recombinant antibody, * = Monoclonal antibody, NA = Not available. Table 3. Table of secondary antibodies used. Company Secondary antibody Catalog number RRID (Antibody Registry) Clonality Concentration (μg/μL) Working concentration (μg/mL) Thermo Fisher Scientific HRP-Goat Anti-Rabbit Antibody (H+L) 65-6120 AB_2533967 polyclonal 1.0 0.2 Thermo Fisher Scientific HRP-Goat Anti-Mouse Antibody (H+L) 62-6520 AB_2533947 polyclonal 1.5 0.75 Millipore Sigma Protein A, HRP conjugate P8651 NA polyclonal - 0.3 Thermo Fisher Scientific Alexa Fluor™ 555-Goat anti-Rabbit IgG (H+L) A-21429 AB_2535850 polyclonal 2.0 0.5 Thermo Fisher Scientific Alexa Fluor™ 555-Goat anti-Mouse IgG (H+L) A-21424 AB_141780 polyclonal 2.0 0.5 Table 4. Illustrations to assess antibody performance in all western blot, immunoprecipitation and immunofluorescence. Western blot Immunoprecipitation Immunofluorescence Results and discussion Our standard protocol involves comparing readouts from wild type (WT) and KO cells. 7 , 8 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. 9 The HAP1 expresses the HNRNPA2B1 transcript at 1.01 log 2 TPM+1. A HNRNPA2B1 KO HAP1 cell line was obtained from Horizon Discovery ( Table 1 ). Moreover, as seen on DepMap, the HAP1 does not carry mutations in the HNRNPA2B1 that could affect antibody–epitope binding. To screen all eight by western blot, WT and HNRNPA2B1 KO protein lysates were ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with eight hnRNP A2/B1 antibodies in parallel ( Figure 1 ). Figure 1. hnRNP A2/B1 antibody screening by western blot. Lysates of HAP1 WT and HNRNPA2B1 KO were prepared, and 30 μg of protein were processed for western blot with the indicated hnRNP A2/B1 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency of acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Antibody dilution used: NB120-6102* at 1/1000; NBP2-80777* at 1/1000; 9304* at 1/1000; GTX114475 at 1/1000; GTX127928 at 1/1000; GTX637305** at 1/1000; 67445-1-Ig* at 1/1000; PA5-81963 at 1/1000. Predicted band size: 37 kDa. *=monoclonal antibody, **=recombinant antibody. We then assessed the capability of all eight antibodies to capture hnRNP A2/B1 from HAP1 protein extracts using immunoprecipitation techniques, followed by western blot analysis. For the immunoblot step, a specific hnRNP A2/B1 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. hnRNP A2/B1 antibody screening by immunoprecipitation. HAP1 WT lysates were prepared, and immunoprecipitation was performed using 2 mg of lysate and 2.0 μg of the indicated hnRNP A2/B1 antibodies pre-coupled to Dynabeads protein A or protein G. Samples were washed and processed for western blot with the hnRNP A2/B1 antibody NBP2-80777* was used at 1/1000. The Ponceau stained transfers of each blot are shown. SM = 4% starting material; UB = 4% unbound fraction; IP = immunoprecipitate, HC = antibody heavy chain. **= recombinant antibody, *= monoclonal antibody. For immunofluorescence, eight antibodies were screened using a mosaic strategy. First, HAP1 WT and HNRNPA2B1 KO cells were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the hnRNP A2/B1 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. 5 Figure 3. hnRNP A2/B1 antibody screening by immunofluorescence. HAP1 WT and HNRNPA2B1 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 hnRNP A2/B1 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 blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, 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: NB120-6102* at 1/1000; NBP2-80777* at 1/1000; 9304* at 1/1000; GTX114475 at 1/1000; GTX127928 at 1/1000; GTX637305** at 1/1000;67445-1-Ig* at 1/800; PA5-81963 at 1/100. Bars = 10 μm. *= monoclonal antibody, **= recombinant antibody. In conclusion, we have screened eight hnRNP A2/B1 commercial antibodies by western blot, immunoprecipitation, and immunofluorescence by comparing the signal produced by the antibodies in human HAP1 WT and HNRNPA2B1 KO cells. To assist users in interpreting antibody performanyce, Table 4 outlines various scenarios in which antibodies may perform in all three applications. 9 High-quality and renewable antibodies that successfully detect hnRNP A2/B1 were identified in all applications. Researchers who wish to study hnRNP A2/B1 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. 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 hnRNP A2/B1 antibodies. YCharOS partners provide approximately 80% of all renewable antibodies, but some top-cited polyclonal antibodies may not be available through these partners. We encourage readers to consult vendor documentation to identify the specific antigen each antibody is raised against, where such information is available. 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 hnRNP A2/B1, only a brief overview of the protein's function and its relevance in disease is provided. hnRNP A2/B1 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. 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 Protocols.io ( protocols.io/view/a-consensus-platform-for-antibody-characterization ). 5 Brief descriptions of the experimental setup used to carry out this study can be found below. Cell lines and antibodies The cell lines, primary and secondary antibodies used in this study are listed in Table 1 , 2 , and 3 , respectively. To ensure consistency with manufacturer recommendations and account for proprietary formulations (where antibody concentrations are not disclosed), antibody usage is reported as dilution ratios rather than absolute concentrations. To facilitate proper citation and unambiguous identification, all cell lines and antibodies are referenced with their corresponding Research Resource Identifiers (RRIDs). 10 , 11 HAP1 KO clone corresponding to the HNRNPA2B1 was purchased from Horizon Discovery commercially. All cell lines used in this study were regularly tested for mycoplasma contamination and were confirmed to be mycoplasma-free. Antibody screening by western blot HAP1 WT and HNRNPA2B1 KO cells were collected in RIPA buffer (25mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Thermo Fisher Scientific, cat. number 89901) supplemented with 1x protease inhibitor cocktail mix (MilliporeSigma, cat. number P8340). Lysates were sonicated briefly and incubated 30 min on ice. Lysates were spun at ~110,000 x 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 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. HAP1 WT lysates 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 x 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 precast midi 4-20% Tris-Glycine polyacrylamide gels. Protein A:HRP was used as a secondary detection system at a concentration of 0.3 μg/ml. Antibody screening by immunofluorescence HAP1 WT and HNRNPA2B1 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°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 pr hnRNP A2/B1 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 x 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. 12 Figures were assembled with Adobe Illustrator. Author contributions Vera Ruíz Moleón-Investigation Riham Ayoubi-Investigation, Supervision, Writing – Original Draft Preparation Maryam Fotouhi-Investigation Joel Ryan-Investigation Donovan Worrall-Investigation Thomas M. Durcan-Investigation Claire M. Brown-Investigation Vincent Francis-Writing – Review & Editing Peter S. McPherson Roles- Supervised and Funding Acquisition Carl Laflamme- Roles: Conceptualization, Funding Acquisition, Writing – Review & Editing All authors reviewed and approved the final version of the manuscript. Data availability Underlying data Zenodo: Dataset for the hnRNP A2/B1 antibody screening study, https://doi.org/10.5281/zenodo.17144167 . 13 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. Liu Y, Shi SL: The roles of hnRNP A2/B1 in RNA biology and disease. Wiley Interdiscip. Rev. RNA. 2021; 12 (2): e1612. Publisher Full Text 2. Lo J, et al. : The RNA binding protein HNRNPA2B1 regulates RNA abundance and motor protein activity in neurites. bioRxiv. 2024. 3. Kim HJ, et al. : Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS. Nature. 2013; 495 (7442): 467–473. PubMed Abstract | Publisher Full Text | Free Full Text 4. Duan Y, et al. : PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins. Cell Res. 2019; 29 (3): 233–247. PubMed Abstract | Publisher Full Text | Free Full Text 5. Ayoubi R, et al. : A consensus platform for antibody characterization. Nat. Protoc. 2024. 6. Biddle MS, Virk HS: YCharOS open antibody characterisation data: Lessons learned and progress made. F1000Res. 2023; 12 : 1344. PubMed Abstract | Publisher Full Text | Free Full Text 7. Laflamme C, et al. : Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. elife. 2019; 8 . PubMed Abstract | Publisher Full Text | Free Full Text 8. Alshafie W, 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 9. Ayoubi R, et al. : Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications. elife. 2023; 12 . Publisher Full Text 10. Bandrowski A, 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 11. 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 12. Stringer C, et al. : Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods. 2021; 18 (1): 100–106. PubMed Abstract | Publisher Full Text 13. Laflamme C: Dataset for hnRNP A2/B1 antibody screening study. [Data set]. Zenodo. 2023. Publisher Full Text Comments on this article Comments (0) Version 1 VERSION 1 PUBLISHED 23 Oct 2025 ADD YOUR COMMENT Comment Author details Author details 1 Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital,Structural Genomics Consortium, ABIF consortium, Montreal, Québec, Canada 2 McGill University, Montreal, Québec, Canada Vera Ruíz Moleón Roles: Investigation Riham Ayoubi Roles: Investigation, Supervision, Writing – Original Draft Preparation Charles Alende Roles: Investigation Maryam Fothouhi Roles: Investigation Joel Ryan Roles: Investigation Sara González Bolívar Roles: Investigation Donovan Worrall Roles: Investigation Thomas M Durcan Roles: Investigation Claire M Brown Roles: Investigation Vincent Francis Roles: Writing – Original Draft Preparation, Writing – Review & Editing Peter S. McPherson Roles: Funding Acquisition, Supervision Carl Laflamme Roles: Conceptualization, Funding Acquisition, Writing – Review & Editing Competing interests For this project, the authors developed partnerships with leading antibody manufacturers and KO cell line providers. The partners provide antibodies and KO cell lines to this project at no cost. These partners include: Abcam, ABCD antibodies, ABclonal, Addgene, Aviva Systems Biology, BioTechne, Cell Signaling Technology, Developmental Studies Hybridoma Bank, GeneTex, Horizon Discovery, Institute for Protein Innovation, MilliporeSigma, Proteintech, Synaptic Systems, Thermo Fisher Scientific. Grant information This work was supported in part by a grant from the ALS-Reproducible Antibody Platform (ALS-RAP). ALS-RAP was created as a public-private partnership by three leading ALS charities: the ALS Association (USA), the Motor Neurone Disease Association (UK), and the ALS Society of Canada. This work was also supported by the Government of Canada through Genome Canada, Genome Quebec, and Ontario Genomics (grant no. OGI-210). 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: 23 Oct 2025, 14:1158 https://doi.org/10.12688/f1000research.170886.1 Copyright © 2025 Moleón VR 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 Moleón VR, Ayoubi R, Alende C et al. A guide to selecting high-performing antibodies for Heterogeneous nuclear ribonucleoproteins A2/B1(hnRNP A2/B1) (UniProt ID: P22626) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 2 approved] . F1000Research 2025, 14 :1158 ( https://doi.org/10.12688/f1000research.170886.1 ) 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 23 Oct 2025 Views 0 Cite How to cite this report: Evans SE. Reviewer Report For: A guide to selecting high-performing antibodies for Heterogeneous nuclear ribonucleoproteins A2/B1(hnRNP A2/B1) (UniProt ID: P22626) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 2 approved] . F1000Research 2025, 14 :1158 ( https://doi.org/10.5256/f1000research.188398.r427059 ) The direct URL for this report is: https://f1000research.com/articles/14-1158/v1#referee-response-427059 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 01 Dec 2025 Sian E. Evans , University of Leicester, Leicester, England, UK Approved VIEWS 0 https://doi.org/10.5256/f1000research.188398.r427059 The article explores the characterisation of eight commercially available antibodies that target Heterogeneous Nuclear Ribonucleoprotein A2/B1 (hnRNP A2/B1). These antibodies have been assessed for their effectiveness in western blotting, immunoprecipitation, and immunofluorescence techniques using HAP1 wild-type (WT) and knockout (KO) ... Continue reading READ ALL The article explores the characterisation of eight commercially available antibodies that target Heterogeneous Nuclear Ribonucleoprotein A2/B1 (hnRNP A2/B1). These antibodies have been assessed for their effectiveness in western blotting, immunoprecipitation, and immunofluorescence techniques using HAP1 wild-type (WT) and knockout (KO) cell lines. The authors include a summary table that provides details about each antibody, including the recommended applications from the vendors for the three techniques mentioned. The methods employed in the study are clearly described and properly cited. Additionally, the inclusion of Table 4, which outlines how to evaluate antibody performance, enhances the data note significantly. 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: Translational research scientist with expertise in developing explant technologies, multi-immunofluorescence, imaging, and histology techniques, as well as molecular biology methods like western blotting and immunoprecipitation. 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 Evans SE. Reviewer Report For: A guide to selecting high-performing antibodies for Heterogeneous nuclear ribonucleoproteins A2/B1(hnRNP A2/B1) (UniProt ID: P22626) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 2 approved] . F1000Research 2025, 14 :1158 ( https://doi.org/10.5256/f1000research.188398.r427059 ) The direct URL for this report is: https://f1000research.com/articles/14-1158/v1#referee-response-427059 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: Moshinsky D. Reviewer Report For: A guide to selecting high-performing antibodies for Heterogeneous nuclear ribonucleoproteins A2/B1(hnRNP A2/B1) (UniProt ID: P22626) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 2 approved] . F1000Research 2025, 14 :1158 ( https://doi.org/10.5256/f1000research.188398.r427060 ) The direct URL for this report is: https://f1000research.com/articles/14-1158/v1#referee-response-427060 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 Nov 2025 Deborah Moshinsky , Institute for Protein Innovation, Boston, Massachusetts, USA Approved VIEWS 0 https://doi.org/10.5256/f1000research.188398.r427060 The authors tested eight Heterogeneous Nuclear Ribonucleoprotein A2/B1 (hnRNP A2/B1) commercial antibodies for activity by Western Blot, immunoprecipitation, and immunofluorescence. They noted which applications were recommended by the vendor and showed data for the results obtained. This is part ... Continue reading READ ALL The authors tested eight Heterogeneous Nuclear Ribonucleoprotein A2/B1 (hnRNP A2/B1) commercial antibodies for activity by Western Blot, immunoprecipitation, and immunofluorescence. They noted which applications were recommended by the vendor and showed data for the results obtained. This is part of a larger ongoing effort to obtain antibody characterization data on commercially available antibodies to assist researchers in choosing antibodies most likely to work in their system. The standardized protocols are clearly described and referenced. This publication should be useful to researchers in need of good hnRNP A2/B1 antibodies. 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: Director of antibody characterization and validation 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 Moshinsky D. Reviewer Report For: A guide to selecting high-performing antibodies for Heterogeneous nuclear ribonucleoproteins A2/B1(hnRNP A2/B1) (UniProt ID: P22626) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 2 approved] . F1000Research 2025, 14 :1158 ( https://doi.org/10.5256/f1000research.188398.r427060 ) The direct URL for this report is: https://f1000research.com/articles/14-1158/v1#referee-response-427060 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 23 Oct 2025 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 Version 1 23 Oct 25 read read Deborah Moshinsky , Institute for Protein Innovation, Boston, USA Sian E. Evans , University of Leicester, Leicester, UK 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 Evans 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. 01 Dec 2025 | for Version 1 Sian E. Evans , University of Leicester, Leicester, England, UK 0 Views copyright © 2025 Evans 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 (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 article explores the characterisation of eight commercially available antibodies that target Heterogeneous Nuclear Ribonucleoprotein A2/B1 (hnRNP A2/B1). These antibodies have been assessed for their effectiveness in western blotting, immunoprecipitation, and immunofluorescence techniques using HAP1 wild-type (WT) and knockout (KO) cell lines. The authors include a summary table that provides details about each antibody, including the recommended applications from the vendors for the three techniques mentioned. The methods employed in the study are clearly described and properly cited. Additionally, the inclusion of Table 4, which outlines how to evaluate antibody performance, enhances the data note significantly. 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 Translational research scientist with expertise in developing explant technologies, multi-immunofluorescence, imaging, and histology techniques, as well as molecular biology methods like western blotting and immunoprecipitation. 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) Evans SE. Peer Review Report For: A guide to selecting high-performing antibodies for Heterogeneous nuclear ribonucleoproteins A2/B1(hnRNP A2/B1) (UniProt ID: P22626) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 2 approved] . F1000Research 2025, 14 :1158 ( https://doi.org/10.5256/f1000research.188398.r427059) 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/14-1158/v1#referee-response-427059 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Moshinsky D. 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 Nov 2025 | for Version 1 Deborah Moshinsky , Institute for Protein Innovation, Boston, Massachusetts, USA 0 Views copyright © 2025 Moshinsky D. 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 authors tested eight Heterogeneous Nuclear Ribonucleoprotein A2/B1 (hnRNP A2/B1) commercial antibodies for activity by Western Blot, immunoprecipitation, and immunofluorescence. They noted which applications were recommended by the vendor and showed data for the results obtained. This is part of a larger ongoing effort to obtain antibody characterization data on commercially available antibodies to assist researchers in choosing antibodies most likely to work in their system. The standardized protocols are clearly described and referenced. This publication should be useful to researchers in need of good hnRNP A2/B1 antibodies. 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 Director of antibody characterization and validation 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) Moshinsky D. Peer Review Report For: A guide to selecting high-performing antibodies for Heterogeneous nuclear ribonucleoproteins A2/B1(hnRNP A2/B1) (UniProt ID: P22626) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 2 approved] . F1000Research 2025, 14 :1158 ( https://doi.org/10.5256/f1000research.188398.r427060) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. 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