A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence

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Edwards" }, { "@type": "Person", "name": "Rachel J. Harding" }, { "@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": "Huntingtin encodes a 3144 amino acid protein, with a polyglutamine repeat tract at the N-terminus. Expansion of this repeat tract above a pathogenic threshold of 36 repeats is the causative mutation of Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons. Here we have characterized twenty Huntingtin 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/13-922/v2", "name": "A guide to selecting high-performing antibodies for Huntingtin (UniProt..." } } ] } Home Browse A guide to selecting high-performing antibodies for Huntingtin (UniProt... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Fanti R, Ayoubi R, Alende C et al. A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.12688/f1000research.153670.2 ) 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 Revised A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] Rebeka Fanti 1 * , Riham Ayoubi 2 * , Charles Alende https://orcid.org/0009-0005-4611-6134 2 , [...] Maryam Fotouhi 2 , Sara González Bolívar https://orcid.org/0000-0002-4299-8281 2 , Renu Chandrasekaran 1 , Kathleen Southern https://orcid.org/0000-0002-4125-3608 2 , Aled M. Edwards 1 , Rachel J. Harding https://orcid.org/0000-0002-1134-391X 1,3 , Carl Laflamme https://orcid.org/0000-0001-5906-025X 2 , NeuroSGC/YCharOS/EDDU collaborative group , ABIF consortium Rebeka Fanti 1 * , Riham Ayoubi 2 * , [...] Charles Alende https://orcid.org/0009-0005-4611-6134 2 , Maryam Fotouhi 2 , Sara González Bolívar https://orcid.org/0000-0002-4299-8281 2 , Renu Chandrasekaran 1 , Kathleen Southern https://orcid.org/0000-0002-4125-3608 2 , Aled M. Edwards 1 , Rachel J. Harding https://orcid.org/0000-0002-1134-391X 1,3 , Carl Laflamme https://orcid.org/0000-0001-5906-025X 2 , NeuroSGC/YCharOS/EDDU collaborative group , ABIF consortium * Equal contributors PUBLISHED 02 Jan 2025 Author details Author details 1 Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada 2 Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Québec, Canada 3 Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, Canada Rebeka Fanti Roles: Investigation, Validation, Writing – Original Draft Preparation Riham Ayoubi Roles: Data Curation, Investigation, Methodology, Validation, Visualization, Writing – Original Draft Preparation Charles Alende Roles: Investigation, Visualization Maryam Fotouhi Roles: Investigation Sara González Bolívar Roles: Investigation Renu Chandrasekaran Roles: Investigation Kathleen Southern Roles: Writing – Original Draft Preparation, Writing – Review & Editing Aled M. Edwards Roles: Funding Acquisition, Supervision Rachel J. Harding Roles: Supervision, Validation Carl Laflamme Roles: Funding Acquisition, Methodology, Project Administration, Resources, Supervision OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the YCharOS (Antibody Characterization through Open Science) gateway. Abstract Huntingtin encodes a 3144 amino acid protein, with a polyglutamine repeat tract at the N-terminus. Expansion of this repeat tract above a pathogenic threshold of 36 repeats is the causative mutation of Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons. Here we have characterized twenty Huntingtin 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 UniProt ID P42858, HTT, Huntingtin, 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 laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: Abcam, 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 Government of Canada through Genome Canada, Genome Quebec, and Ontario Genomics (grant no. OGI-210). RF and RA are supported by Mitacs fellowships. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 Fanti R 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: Fanti R, Ayoubi R, Alende C et al. A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.12688/f1000research.153670.2 ) First published: 13 Aug 2024, 13 :922 ( https://doi.org/10.12688/f1000research.153670.1 ) Latest published: 02 Jan 2025, 13 :922 ( https://doi.org/10.12688/f1000research.153670.2 ) Revised Amendments from Version 1 An error occurred as only half of the antibodies tested by our team were included in Figure 3. The original submission featured a split Figure 3 with Parts 1 and 2 uploaded separately. The revised Figure 3 now includes all 20 HTT antibodies tested. An error occurred as only half of the antibodies tested by our team were included in Figure 3. The original submission featured a split Figure 3 with Parts 1 and 2 uploaded separately. The revised Figure 3 now includes all 20 HTT antibodies tested. To read any peer review reports and author responses for this article, follow the "read" links in the Open Peer Review table. READ REVIEWER RESPONSES Introduction Huntington’s Disease (HD) is a neurodegenerative disorder inherited in an autosomal dominant manner, presenting with a spectrum of progressive motor, cognitive, and psychological impairments, typically with adult-onset of symptoms. 1 Although the HD causative gene, HTT , was discovered over three decades ago, there are still no disease-modifying treatments available for patients, and progress unpicking the molecular pathology of the disease remains slow. 2 HD arises from a heterozygous expansion mutation of the trinucleotide CAG repeat tract in exon 1 of HTT , located on chromosome 4, above a critical threshold of ~36 repeats. This mutation results in expansion of the polyglutamine stretch at the N-terminus of the 3144 amino acid Huntingtin protein. Huntingtin functions as a scaffold protein, engaging in extensive protein-protein interactions, 3 forming various multi-protein complexes to carry-out its diverse array of functions. Modulation of this interaction network by the polyglutamine expansion contributes to degeneration within the central nervous system, affecting medium spiny neurons at the onset of disease. 4 , 5 The low expression level, complex interactome and large size of the 348 kDa Huntingtin protein have given rise to technical challenges which have hindered precise determination of its molecular function, or how this is altered in disease. In particular, the use of different Huntingtin antibodies by scientists in the HD research community, often mapping to structurally distant epitopes, can yield different or even conflicting results, further conflating interrogation of this protein. 6 – 8 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. 9 – 11 Here we evaluated the performance of twenty commercial antibodies for Huntingtin for use in western blot, immunoprecipitation, and immunofluorescence, enabling biochemical and cellular assessment of Huntingtin properties and function. The platform for antibody characterization used to carry out this study was endorsed by a committee of industry and 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 target protein. The standardized consensus antibody characterization protocols are openly available on Protocol Exchange (DOI: 10.21203/rs.3.pex-2607/v1 ). 12 The authors do not engage in result analysis or offer explicit antibody recommendations. A limitation of this study is the use of universal protocols - any conclusions remain relevant within the confines of the experimental setup and cell line used in this study. 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. 13 Results and discussion Our standard protocol involves comparing readouts from WT (wild type) and KO cells. 14 , 15 The first step is to identify a cell line(s) that expresses sufficient levels of a given protein to generate a measurable signal. To this end, we examined the DepMap 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 (Cancer Dependency Map Portal, RRID:SCR_017655). We generated an HTT KO line in DMS 53 as it expresses the endogenous HTT transcript at 6.1 log 2 (TPM+1), which is above the average range of cancer cell lines analyzed. A commercial HAP1 HTT KO is also available; HAP1 expresses HTT at 3.7 log 2 (TPM+1) RNA level. A HEK293T HTT KO cell line has been developed and used elsewhere 16 ( Table 1 ). All three cell line backgrounds were evaluated by western blot using a high-performing Huntingtin antibody detected in Figure 1 . DMS 53 was identified as the most suitable cell line ( Figure 2 ), which can be explained by its high expression of the HTT transcript compared to other two cell lines. Thus, DMS 53 WT and KO cell lines were generated and used to evaluate the antibodies in all applications. Table 1. Summary of the cell lines used. Institution Catalog number RRID (Cellosaurus) Cell line Genotype ATCC CRL-2062 CVCL_1177 DMS 53 WT Academic non-commercial CVCL_D6U0 DMS 53 HTT KO ATCC CRL-3216 CVCL_0063 HEK 293T WT Academic non-commercial CVCL_D7EP HEK 293T HTT KO 16 Horizon Discovery C631 CVCL_Y019 HAP1 WT Horizon Discovery HZGHC004595c006 CVCL_SR86 HAP1 HTT KO Figure 1. Huntingtin antibody screening by western blot. Lysates of DMS 53 (WT and HTT KO) were prepared and 30 μg of protein were processed for western blot with the indicated Huntingtin antibodies. Tris-Glycine 4-20% gels were used for SDS-PAGE. 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. Exceptions were given for antibodies ab109115**, ab45169**, and MA5-41256** which were titrated as the signal was too weak when following the supplier’s recommendations. Antibody dilution used: ab109115** at 1/2000, ab45169** at 1/5000, ABCD_AG854** at 1/10, ABCD_AG855** at 1/10, A19064** at 1/1000, 5656** at 1/1000, CH03023* at 1/1000, MW1-S* at 1/10, MW3-S* at 1/10, MW4-S* at 1/10, MW5-S* at 1/10, MW6-S* at 1/10, MW7-S* at 1/10, MW8-S* at 1/10, GTX132433 at 1/500, GTX638832** at 1/500, 710695** at 1/200, MA3-040* at 1/1000, MA5-16703* at 1/500, MA5-41256** at 1/2000. Predicted band size: 347 kDa. *Monoclonal antibody, **Recombinant antibody. Note: MW1-S*, MW3-S*, MW4-S*, MW5-S*, MW6-S*, MW7-S* and MW8-S* are expected to only recognize an altered conformation of the polyQ domain generated as the polyQ domain of Huntingtin increases in length. Figure 2. Huntingtin western blot on various cell lysates. Lysates of WT and HTT KO in DMS 53, HEK 293T and HAP1 were prepared, and 30 μg of protein was processed for western blot with the indicated Huntingtin antibodies ab45169** at 1/5000 and GTX638832** at 1/500. Tris-Glycine 4-20% gels were used for SDS-PAGE. The Ponceau stained transfer is shown as a loading control. Predicted band size: 347 kDa. **Recombinant antibody. For western blot experiments, WT and HTT KO protein lysates were ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with twenty Huntingtin antibodies in parallel ( Table 2 , Figure 1 ). Table 2. Summary of the Huntingtin antibodies tested. Company Catalog number Lot number RRID (Antibody Registry) Clonality Clone ID Host Concentration (μg/μl) Vendors recommended applications Abcam ab109115 ** 1003147-4 AB_10863082 recombinant-mono EPR5526 rabbit 1.52 Wb, IF Abcam ab45169 ** 1022500 AB_733062 recombinant-mono EP867Y rabbit 1.55 Wb, IF ABCD Antibodies ABCD_AG854 ** 10/27/2023 AB_3076339 recombinant-mono 12.3 rabbit 0.004 n/a ABCD Antibodies ABCD_AG855 ** 10/27/2023 AB_3076340 recombinant-mono C4 rabbit 0.19 n/a ABclonal A19064 ** 4000000431 AB_2862557 recombinant-mono ARC0431 rabbit 0.63 Wb, IF Cell Signaling Technology 5656 ** 6 AB_10827977 recombinant-mono D7F7 rabbit 0.10 Wb, IF Coriell Institute CH03023 * 03.17.21 AB_3096092 monoclonal 2B7 mouse 1.71 n/a DSHB MW1-S * 44322 AB_528290 monoclonal MW1-S mouse 0.02 Wb, IP, IF DSHB MW3-S * 43216 AB_528292 monoclonal MW3-S mouse 0.01 Wb, IF DSHB MW4-S * 43251 AB_528293 monoclonal MW4-S mouse 0.02 Wb, IF DSHB MW5-S * 44742 AB_528294 monoclonal MW5-S mouse 0.03 Wb, IF DSHB MW6-S * 43230 AB_528295 monoclonal MW6-S mouse 0.06 Wb, IF DSHB MW7-S * 43461 AB_528296 monoclonal MW7-S mouse 0.03 Wb, IF DSHB MW8-S * 44315 AB_528297 monoclonal MW8-S mouse 0.04 Wb, IP, IF GeneTex GTX132433 42312 AB_2886646 polyclonal MW3-S rabbit 1.40 Wb, IF GeneTex GTX638832 ** 45096 AB_3094813 recombinant-mono HL2483 rabbit 0.98 Wb Thermo Fisher Scientific 710695 ** RF236710 AB_2608784 recombinant-poly 3HCLC rabbit 0.50 IF Thermo Fisher Scientific MA3-040 * YG376237 AB_2608783 monoclonal 1HU-4C8 mouse n/a Wb, IF Thermo Fisher Scientific MA5-16703 * YE3913821A AB_2538195 monoclonal HDB4E10 mouse 1.00 Wb, IP, IF Thermo Fisher Scientific MA5-41256 ** YE3913382B AB_2899009 recombinant-mono JB89-34 rabbit 1.00 Wb, IF * Monoclonal antibody, ** Recombinant antibody. We then assessed the capability of all twenty antibodies to capture Huntingtin from DMS 53 protein extracts using immunoprecipitation techniques, followed by western blot analysis. For the immunoblot step, a specific Huntingtin antibody identified previously (refer to Figure 1 ) was selected. Equal amounts of the starting material (SM), the unbound fraction (UB), as well as the whole immunoprecipitate (IP) eluates were separated by SDS-PAGE ( Figure 3 ). Figure 3. Huntingtin antibody screening by immunoprecipitation. DMS 53 lysates were prepared, and immunoprecipitation was performed using 2.0 μg of the indicated Huntingtin antibodies pre-coupled to Dynabeads protein A or protein G. The concentration of MA3-040* is unknown and therefore 5 μL of this antibody was tested. All samples were washed and processed for western blot with the indicated Huntingtin antibody. Tris-Glycine 4-20% gels were used for SDS-PAGE. For western blot, ab45169** was used at 1/5000. The Ponceau stained transfers of each blot are shown. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate. *Monoclonal antibody, **Recombinant antibody. For immunofluorescence, twenty antibodies were screened using a mosaic strategy. First, DMS 53 WT and HTT KO cells were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the Huntingtin antibodies were evaluated. Both WT and KO lines were imaged in the same field of view to reduce staining, imaging and image analysis bias ( Figure 4 ). Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested, 12 and the images presented in Figure 3 are representative of this analysis. Figure 4. Huntingtin antibody screening by immunofluorescence. DMS 53 WT and HTT 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 Huntingtin 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 line, respectively. When an antibody was recommended for immunofluorescence by the supplier, we tested it at the recommended dilution and at 1 μg/ml. The rest of the antibodies were tested at 1 and 2 μg/ml. The final concentration of each antibody was selected based on the detection range of the microscope used and a quantitative analysis not shown here. Antibody dilutions corresponding to the images shown are: ab109115** at 1/1500, ab45169** at 1/1500, ABCD_AG854** at 1/500, ABCD_AG855** at 1/200, A19064** at 1/600, 5656** at 1/100, CH03023* at 1/1700, MW1-S* at 1/20, MW3-S* at 1/10, MW4-S* at 1/10, MW5-S* at 1/15, MW6-S* at 1/60, MW7-S* at 1/15, MW8-S* at 1/20, GTX132433 at 1/500, GTX638832** at 1/500, 710695** at 1/500, MA3-040* at 1/1000, MA5-16703* at 1/1000, MA5-41256** at 1/1000. Bars = 10 μm. *Monoclonal antibody, **Recombinant antibody. In conclusion, we have screened twenty commercial Huntingtin antibodies by western blot, immunoprecipitation, and immunofluorescence by comparing the signal produced by the antibodies in human DMS 53 WT and HTT KO cells. Several high-quality and renewable Huntingtin antibodies were identified in all applications. Researchers who wish to study Huntingtin 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. 17 Methods 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 ). 12 Antibodies and cell lines used 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. 18 , 19 Data availability Underlying data Zenodo: Dataset for the Huntingtin antibody screening study, doi.org/10.5281/zenodo.11639052 . 17 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 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. An earlier version of this of this article can be found on Zenodo (DOI: 10.5281/zenodo.11582780 ). References 1. Ross CA, Tabrizi SJ: Huntington's disease: from molecular pathogenesis to clinical treatment. Lancet Neurol. 2011; 10 (1): 83–98. Publisher Full Text 2. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The Huntington's Disease Collaborative Research Group. Cell. 1993; 72 (6): 971–983. PubMed Abstract | Publisher Full Text 3. Greco TM, Secker C, Ramos ES, et al. : Dynamics of huntingtin protein interactions in the striatum identifies candidate modifiers of Huntington disease. Cell Syst. 2022; 13 (4): 304–320.e5. PubMed Abstract | Publisher Full Text | Free Full Text 4. Matlik K, Baffuto M, Kus L, et al. : Cell-type-specific CAG repeat expansions and toxicity of mutant Huntingtin in human striatum and cerebellum. Nat. Genet. 2024; 56 (3): 383–394. PubMed Abstract | Publisher Full Text | Free Full Text 5. Pressl C, Matlik K, Kus L, et al. : Selective vulnerability of layer 5a corticostriatal neurons in Huntington's disease. Neuron. 2024; 112 (6): 924–941.e10. PubMed Abstract | Publisher Full Text 6. Strong TV, Tagle DA, Valdes JM, et al. : Widespread expression of the human and rat Huntington's disease gene in brain and nonneural tissues. Nat. Genet. 1993; 5 (3): 259–265. PubMed Abstract | Publisher Full Text 7. Greco TM, Secker C, Ramos ES, et al. : Dynamics of huntingtin protein interactions in the striatum identifies candidate modifiers of Huntington disease. Cell Syst. 2022; 13 (4): 304–20.e5. PubMed Abstract | Publisher Full Text | Free Full Text 8. Wanker EE, Ast A, Schindler F, et al. : The pathobiology of perturbed mutant huntingtin protein-protein interactions in Huntington's disease. J. Neurochem. 2019; 151 (4): 507–519. PubMed Abstract | Publisher Full Text 9. 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 10. 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 11. 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 12. Ayoubi R, Ryan J, Bolivar SG, et al. : A consensus platform for antibody characterization (Version 1). Protocol. Exchange. 2024. Publisher Full Text 13. Biddle MS, Virk HS: YCharOS open antibody characterisation data: Lessons learned and progress made. F1000Res. 2023; 12 (12): 1344. Publisher Full Text 14. 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 15. 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 16. Jung R, Lee Y, Barker D, et al. : Mutations causing Lopes-Maciel-Rodan syndrome are huntingtin hypomorphs. Hum. Mol. Genet. 2021; 30 (3-4): 135–148. PubMed Abstract | Publisher Full Text | Free Full Text 17. Ayoubi R, Laflamme C: Dataset for the Huntingtin antibody screening study. [Data set]. Zenodo. 2024. 18. 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 19. 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 Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 13 Aug 2024 ADD YOUR COMMENT Comment Author details Author details 1 Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada 2 Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Québec, Canada 3 Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, Canada Rebeka Fanti Roles: Investigation, Validation, Writing – Original Draft Preparation Riham Ayoubi Roles: Data Curation, Investigation, Methodology, Validation, Visualization, Writing – Original Draft Preparation Charles Alende Roles: Investigation, Visualization Maryam Fotouhi Roles: Investigation Sara González Bolívar Roles: Investigation Renu Chandrasekaran Roles: Investigation Kathleen Southern Roles: Writing – Original Draft Preparation, Writing – Review & Editing Aled M. Edwards Roles: Funding Acquisition, Supervision Rachel J. Harding Roles: Supervision, Validation Carl Laflamme Roles: Funding Acquisition, Methodology, Project Administration, Resources, Supervision Competing interests For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: Abcam, 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 Government of Canada through Genome Canada, Genome Quebec, and Ontario Genomics (grant no. OGI-210). RF and RA are supported by Mitacs fellowships. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (2) version 2 Revised Published: 02 Jan 2025, 13:922 https://doi.org/10.12688/f1000research.153670.2 version 1 Published: 13 Aug 2024, 13:922 https://doi.org/10.12688/f1000research.153670.1 Copyright © 2025 Fanti R 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 Fanti R, Ayoubi R, Alende C et al. A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.12688/f1000research.153670.2 ) 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 2 VERSION 2 PUBLISHED 02 Jan 2025 Revised Views 0 Cite How to cite this report: Bertoglio D. Reviewer Report For: A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.176211.r360581 ) The direct URL for this report is: https://f1000research.com/articles/13-922/v2#referee-response-360581 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 12 Feb 2025 Daniele Bertoglio , University of Antwerp, Antwerp, Belgium Approved VIEWS 0 https://doi.org/10.5256/f1000research.176211.r360581 This manuscript by Fanti et al. presents a comprehensive evaluation of twenty commercially available HTT antibodies utilizing standardized experimental protocols. The study systematically assesses the suitability and sensitivity of these antibodies in Western blot, immunoprecipitation, and immunofluorescence applications, employing both ... Continue reading READ ALL This manuscript by Fanti et al. presents a comprehensive evaluation of twenty commercially available HTT antibodies utilizing standardized experimental protocols. The study systematically assesses the suitability and sensitivity of these antibodies in Western blot, immunoprecipitation, and immunofluorescence applications, employing both wild-type and knock-out HTT cell lines. It would have been of additional support inclusion of immunohistochemistry, but this does not restrict the value of the work. The study addresses a critical technical challenge in Huntington’s Disease (HD) research, namely the variability in antibody performance, which has posed a significant issue within the field. The experimental approach is well conceived, designed, and executed. The data are presented in a clear and accessible manner, facilitating ease of interpretation. The findings provide valuable guidance for researchers seeking to source and utilize specific HTT antibodies in their investigations. Overall, this study constitutes a significant contribution to the HD research community, offering essential data to improve experimental consistency and reproducibility 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: In vivo and post-mortem imaging of neurodegenetive disorders, including HD 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 Bertoglio D. Reviewer Report For: A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.176211.r360581 ) The direct URL for this report is: https://f1000research.com/articles/13-922/v2#referee-response-360581 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: Subramaniam S. Reviewer Report For: A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.176211.r358661 ) The direct URL for this report is: https://f1000research.com/articles/13-922/v2#referee-response-358661 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 06 Feb 2025 Srinivasa Subramaniam , The Scripps Research Institute, Jupiter, USA Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.176211.r358661 Fanti et al. evaluated twenty commercial antibodies against huntingtin for Western blot, immunoprecipitation, and immunofluorescence by employing a defined experimental approach that involved comparing results in deletion cell lines with isogenic parental controls. This study is a crucial guide that ... Continue reading READ ALL Fanti et al. evaluated twenty commercial antibodies against huntingtin for Western blot, immunoprecipitation, and immunofluorescence by employing a defined experimental approach that involved comparing results in deletion cell lines with isogenic parental controls. This study is a crucial guide that will significantly enhance research on huntingtin biology and Huntington's disease. The authors employed twenty commercial antibodies and three distinct cell lines, validating the antibodies across several assays. I have a question concerning IP. It is unclear whether the authors employed control beads as an extra control measure during the immunoprecipitation process. Figure 3 necessitates either control beads or immunoprecipitation conducted in knockout cells. 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: Mechanisms of neurodegenerative disease 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 Subramaniam S. Reviewer Report For: A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.176211.r358661 ) The direct URL for this report is: https://f1000research.com/articles/13-922/v2#referee-response-358661 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: Saiyin H. Reviewer Report For: A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.176211.r360585 ) The direct URL for this report is: https://f1000research.com/articles/13-922/v2#referee-response-360585 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 06 Feb 2025 Hexige Saiyin , Fudan University, Shanghai, China Approved VIEWS 0 https://doi.org/10.5256/f1000research.176211.r360585 This well-designed study validates twenty HTT antibodies by WB and IF. Based on the reviewer's experience and other studies, HTT is unstable after fixation in human tissue and cells (Ferrante, Gutekunst et al. 1997)(ref 2). Thus, the reviewer suggests providing ... Continue reading READ ALL This well-designed study validates twenty HTT antibodies by WB and IF. Based on the reviewer's experience and other studies, HTT is unstable after fixation in human tissue and cells (Ferrante, Gutekunst et al. 1997)(ref 2). Thus, the reviewer suggests providing the details of the fixation in IF and processing the WB sample in WB, especially the staining/blotting time after the fixation or harvesting WB (such as the staining performed immediately after fixation or 48 hours after fixation et al.). Minor concerns: The resolution of IF images is low. The cellular distribution patterns of HTT in cells are difficult to read. Thus, the reviewer encourages showing high-resolution images in IF, which can provide more detail of cellular distribution patterns. HTTs are easy to aggregate (DiFiglia, Sapp et al. 1997, Gutekunst, Li et al. 1999)(ref 1 and 3). The author only detected the single HTT but not the aggregate. Does the antibody that recognizes the single HTT also equally detect the aggregate of cells or tissue? Does the author try to detect the aggregate by those antibodies? Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Partly Are sufficient details of methods and materials provided to allow replication by others? Partly Are the datasets clearly presented in a useable and accessible format? Yes References 1. DiFiglia M, Sapp E, Chase KO, Davies SW, et al.: Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science . 1997; 277 (5334): 1990-3 PubMed Abstract | Publisher Full Text 2. Ferrante RJ, Gutekunst CA, Persichetti F, McNeil SM, et al.: Heterogeneous topographic and cellular distribution of huntingtin expression in the normal human neostriatum. J Neurosci . 1997; 17 (9): 3052-63 PubMed Abstract | Publisher Full Text 3. Gutekunst CA, Li SH, Yi H, Mulroy JS, et al.: Nuclear and neuropil aggregates in Huntington's disease: relationship to neuropathology. J Neurosci . 1999; 19 (7): 2522-34 PubMed Abstract | Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: Pathogenesis of HD and 3D histology and pathology of human disease. 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 Saiyin H. Reviewer Report For: A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.176211.r360585 ) The direct URL for this report is: https://f1000research.com/articles/13-922/v2#referee-response-360585 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: Landles C. Reviewer Report For: A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.176211.r360584 ) The direct URL for this report is: https://f1000research.com/articles/13-922/v2#referee-response-360584 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 Christian Landles , University College London, London, UK Approved VIEWS 0 https://doi.org/10.5256/f1000research.176211.r360584 This manuscript by Fanti et al. has comprehensively characterized twenty commercially available HTT antibodies using cell lines which were either wild-type or knock-out for the HTT gene. HTT antibody suitability and sensitivity was assessed using standardized experimental protocols using the ... Continue reading READ ALL This manuscript by Fanti et al. has comprehensively characterized twenty commercially available HTT antibodies using cell lines which were either wild-type or knock-out for the HTT gene. HTT antibody suitability and sensitivity was assessed using standardized experimental protocols using the applications of western blot, immunoprecipitation, and immunofluorescence. This experimental approach was rationally conceived and executed, and the experimental dataset has been presented in a clear manner which was easy to follow and interpret. Overall, this data will be of great use should anyone within the Huntington’s disease research community wish to source and try any specific HTT antibody for their own research needs. Minor Comments and Recommendations Introduction: The expression level of the full-length HTT protein is not particularly “low” in brain tissues, it is peripheral tissues which express full-length HTT and low levels. Results and discussion: Table 1, it would be useful to report what lineage these cell lines used are from, and also state the CAG repeat size of these cell lines. The MW1 to MW7 antibodies, originally characterized in Ko, et al ., Brain Res. Bull., 2001, predominantly recognise mutant HTT isoforms where the pathogenic CAG tract has been expanded. Therefore, although stated in the figure legend of Fig. 1 “MW1-S*, MW3-S*, MW4-S*, MW5-S*, MW6-S*, MW7-S* and MW8-S* are expected to only recognize an altered conformation of the polyQ domain generated as the polyQ domain of Huntingtin increases in length.”, I am not sure why these antibodies were being tested here on wild-type DMS 53 cell lines where there was no expanded CAG mutation. A negative signal here was always to be expected. The MW8 antibody, originally characterized in Ko, et al ., Brain Res. Bull., 2001 [ref 1], and later mapped by Landles et al ., JBC, 2010 [ref 2], has been demonstrated to be a neo-epitope antibody specifically recognizing the highly pathogenic mutant HTT1a protein isoform. It has subsequently been shown in numerous research papers to not recognise the full-length HTT protein isoform, and so likewise, a negative signal here on wild-type DMS 53 cells was always to be expected. (It would be helpful if these two points were explained fully in the main body of text and not buried in the figure legend of Fig. 1). Since Fanti et al. was comprehensively characterizing commercial HTT antibodies, I was very surprised that the commercial monoclonal HTT-MAB5490 and HTT-MAB2166 antibodies were used in their analyses. These two monoclonal HTT antibodies are widely used throughout the entire Huntington’s disease research community, and characterising these here (maybe instead of the MW1-MW8 series) would have been more appropriate / valuable. Methods: In the interest of having all the methodologies used in this already concise manuscript, it would be helpful to the reader to have all the experimental methodologies within this section, as opposed to viewing them from another link online. 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? Partly Are the datasets clearly presented in a useable and accessible format? Yes References 1. Ko J, Ou S, Patterson PH: New anti-huntingtin monoclonal antibodies: implications for huntingtin conformation and its binding proteins. Brain Res Bull . 56 (3-4): 319-29 PubMed Abstract | Publisher Full Text 2. Landles C, Sathasivam K, Weiss A, Woodman B, et al.: Proteolysis of mutant huntingtin produces an exon 1 fragment that accumulates as an aggregated protein in neuronal nuclei in Huntington disease. J Biol Chem . 2010; 285 (12): 8808-23 PubMed Abstract | Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: The molecular pathogenesis of Huntingtin's disease, pre-clinical Huntington's disease mouse models, HTT protein bioassays, Molecular biology, HTT protein aggregation. 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 Landles C. Reviewer Report For: A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.176211.r360584 ) The direct URL for this report is: https://f1000research.com/articles/13-922/v2#referee-response-360584 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 Version 1 VERSION 1 PUBLISHED 13 Aug 2024 Views 0 Cite How to cite this report: Bowman K. Reviewer Report For: A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.168592.r315013 ) The direct URL for this report is: https://f1000research.com/articles/13-922/v1#referee-response-315013 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 09 Sep 2024 Karen Bowman , University of Leicester, Leicester, England, UK Approved VIEWS 0 https://doi.org/10.5256/f1000research.168592.r315013 The article pointed out the current technical difficulties in studying Huntington’s Disease and identified the differences in antibodies to the protein Huntingtin, used throughout the HD research community, as a particular problem. The study formed part of the wider YCharOS ... Continue reading READ ALL The article pointed out the current technical difficulties in studying Huntington’s Disease and identified the differences in antibodies to the protein Huntingtin, used throughout the HD research community, as a particular problem. The study formed part of the wider YCharOS collaboration to characterise commercial antibodies for human proteins using standardized protocols with the intention of improving antibody reproducibility issues. The study was an evaluation of twenty commercial antibodies for the protein Huntingtin for use in western blot, immunoprecipitation, and immunofluorescence. The study was well designed and the results were to a high technical standard and quality. The data is presented clearly and looks robust. It was reassuring to see that a committee of industry and academic representatives had endorsed the platform used. The platform consisted of identification of human cell lines suitable for antibody characterisation studies i.e. with adequate levels of Huntingtin, followed by the development of and contribution of isogenic knockout control cell lines, which were validated at the protein level. The final step was a series of antibody characterisation procedures, limited to the most common research uses of antibodies. Limitations of the study are clearly stated. Two minor errors were spotted. There is an inconsistency between the text and figure 3, where they mention IP was done on twenty antibodies in the text, but results for only ten antibodies are presented in the figure. Perhaps some additional text on why only ten were presented would be of use, or amend the text to reflect what is presented in the figure. A second error is that in the final results paragraph on immunofluorescence, figure 3 is cited, and linked back to, instead of figure 4. Overall, this will be a highly useful resource for scientists in the HD research community. 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: drug discovery and diagnostics 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 Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.168592.r315013 ) The direct URL for this report is: https://f1000research.com/articles/13-922/v1#referee-response-315013 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: Shao J. Reviewer Report For: A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.168592.r315014 ) The direct URL for this report is: https://f1000research.com/articles/13-922/v1#referee-response-315014 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 28 Aug 2024 Jieya Shao , Washington University in St Louis, St. Louis, Missouri, USA Approved VIEWS 0 https://doi.org/10.5256/f1000research.168592.r315014 In this study, the authors performed comprehensive analysis of twenty commercially available HTT antibodies for their suitability and performance in Western blot, immunoprecipitation, and immunofluorescence staining applications. The experimental approaches were logically and thoroughly designed. Data are of high-quality and ... Continue reading READ ALL In this study, the authors performed comprehensive analysis of twenty commercially available HTT antibodies for their suitability and performance in Western blot, immunoprecipitation, and immunofluorescence staining applications. The experimental approaches were logically and thoroughly designed. Data are of high-quality and clearly presented. The results will be very helpful for researchers in the HTT field. However, there seems to be two small errors. First, in Figure 3, only the IP data of 10 antibodies were shown while in the text it was stated that 20 antibodies were analyzed. Second, on Page 5, in the last sentence of the second paragraph, the authors seem to cite Figure 3 by mistake. 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 Shao J. Reviewer Report For: A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.168592.r315014 ) The direct URL for this report is: https://f1000research.com/articles/13-922/v1#referee-response-315014 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 2 VERSION 2 PUBLISHED 13 Aug 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 5 6 Version 2 (revision) 02 Jan 25 read read read read Version 1 13 Aug 24 read read Jieya Shao , Washington University in St Louis, St. Louis, USA Karen Bowman , University of Leicester, Leicester, UK Christian Landles , University College London, London, UK Hexige Saiyin , Fudan University, Shanghai, China Srinivasa Subramaniam , The Scripps Research Institute, Jupiter, USA Daniele Bertoglio , University of Antwerp, Antwerp, Belgium 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 Bertoglio 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. 12 Feb 2025 | for Version 2 Daniele Bertoglio , University of Antwerp, Antwerp, Belgium 0 Views copyright © 2025 Bertoglio 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 This manuscript by Fanti et al. presents a comprehensive evaluation of twenty commercially available HTT antibodies utilizing standardized experimental protocols. The study systematically assesses the suitability and sensitivity of these antibodies in Western blot, immunoprecipitation, and immunofluorescence applications, employing both wild-type and knock-out HTT cell lines. It would have been of additional support inclusion of immunohistochemistry, but this does not restrict the value of the work. The study addresses a critical technical challenge in Huntington’s Disease (HD) research, namely the variability in antibody performance, which has posed a significant issue within the field. The experimental approach is well conceived, designed, and executed. The data are presented in a clear and accessible manner, facilitating ease of interpretation. The findings provide valuable guidance for researchers seeking to source and utilize specific HTT antibodies in their investigations. Overall, this study constitutes a significant contribution to the HD research community, offering essential data to improve experimental consistency and reproducibility 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 In vivo and post-mortem imaging of neurodegenetive disorders, including HD 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) Bertoglio D. Peer Review Report For: A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.176211.r360581) 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/13-922/v2#referee-response-360581 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Subramaniam 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. 06 Feb 2025 | for Version 2 Srinivasa Subramaniam , The Scripps Research Institute, Jupiter, USA 0 Views copyright © 2025 Subramaniam 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 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 Fanti et al. evaluated twenty commercial antibodies against huntingtin for Western blot, immunoprecipitation, and immunofluorescence by employing a defined experimental approach that involved comparing results in deletion cell lines with isogenic parental controls. This study is a crucial guide that will significantly enhance research on huntingtin biology and Huntington's disease. The authors employed twenty commercial antibodies and three distinct cell lines, validating the antibodies across several assays. I have a question concerning IP. It is unclear whether the authors employed control beads as an extra control measure during the immunoprecipitation process. Figure 3 necessitates either control beads or immunoprecipitation conducted in knockout cells. 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 Mechanisms of neurodegenerative disease 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) Subramaniam S. Peer Review Report For: A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.176211.r358661) 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/13-922/v2#referee-response-358661 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Saiyin H. 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. 06 Feb 2025 | for Version 2 Hexige Saiyin , Fudan University, Shanghai, China 0 Views copyright © 2025 Saiyin H. 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 This well-designed study validates twenty HTT antibodies by WB and IF. Based on the reviewer's experience and other studies, HTT is unstable after fixation in human tissue and cells (Ferrante, Gutekunst et al. 1997)(ref 2). Thus, the reviewer suggests providing the details of the fixation in IF and processing the WB sample in WB, especially the staining/blotting time after the fixation or harvesting WB (such as the staining performed immediately after fixation or 48 hours after fixation et al.). Minor concerns: The resolution of IF images is low. The cellular distribution patterns of HTT in cells are difficult to read. Thus, the reviewer encourages showing high-resolution images in IF, which can provide more detail of cellular distribution patterns. HTTs are easy to aggregate (DiFiglia, Sapp et al. 1997, Gutekunst, Li et al. 1999)(ref 1 and 3). The author only detected the single HTT but not the aggregate. Does the antibody that recognizes the single HTT also equally detect the aggregate of cells or tissue? Does the author try to detect the aggregate by those antibodies? Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Partly Are sufficient details of methods and materials provided to allow replication by others? Partly Are the datasets clearly presented in a useable and accessible format? Yes References 1. DiFiglia M, Sapp E, Chase KO, Davies SW, et al.: Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science . 1997; 277 (5334): 1990-3 PubMed Abstract | Publisher Full Text 2. Ferrante RJ, Gutekunst CA, Persichetti F, McNeil SM, et al.: Heterogeneous topographic and cellular distribution of huntingtin expression in the normal human neostriatum. J Neurosci . 1997; 17 (9): 3052-63 PubMed Abstract | Publisher Full Text 3. Gutekunst CA, Li SH, Yi H, Mulroy JS, et al.: Nuclear and neuropil aggregates in Huntington's disease: relationship to neuropathology. J Neurosci . 1999; 19 (7): 2522-34 PubMed Abstract | Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise Pathogenesis of HD and 3D histology and pathology of human disease. 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) Saiyin H. Peer Review Report For: A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.176211.r360585) 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/13-922/v2#referee-response-360585 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Landles C. 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 2 Christian Landles , University College London, London, UK 0 Views copyright © 2025 Landles C. 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 This manuscript by Fanti et al. has comprehensively characterized twenty commercially available HTT antibodies using cell lines which were either wild-type or knock-out for the HTT gene. HTT antibody suitability and sensitivity was assessed using standardized experimental protocols using the applications of western blot, immunoprecipitation, and immunofluorescence. This experimental approach was rationally conceived and executed, and the experimental dataset has been presented in a clear manner which was easy to follow and interpret. Overall, this data will be of great use should anyone within the Huntington’s disease research community wish to source and try any specific HTT antibody for their own research needs. Minor Comments and Recommendations Introduction: The expression level of the full-length HTT protein is not particularly “low” in brain tissues, it is peripheral tissues which express full-length HTT and low levels. Results and discussion: Table 1, it would be useful to report what lineage these cell lines used are from, and also state the CAG repeat size of these cell lines. The MW1 to MW7 antibodies, originally characterized in Ko, et al ., Brain Res. Bull., 2001, predominantly recognise mutant HTT isoforms where the pathogenic CAG tract has been expanded. Therefore, although stated in the figure legend of Fig. 1 “MW1-S*, MW3-S*, MW4-S*, MW5-S*, MW6-S*, MW7-S* and MW8-S* are expected to only recognize an altered conformation of the polyQ domain generated as the polyQ domain of Huntingtin increases in length.”, I am not sure why these antibodies were being tested here on wild-type DMS 53 cell lines where there was no expanded CAG mutation. A negative signal here was always to be expected. The MW8 antibody, originally characterized in Ko, et al ., Brain Res. Bull., 2001 [ref 1], and later mapped by Landles et al ., JBC, 2010 [ref 2], has been demonstrated to be a neo-epitope antibody specifically recognizing the highly pathogenic mutant HTT1a protein isoform. It has subsequently been shown in numerous research papers to not recognise the full-length HTT protein isoform, and so likewise, a negative signal here on wild-type DMS 53 cells was always to be expected. (It would be helpful if these two points were explained fully in the main body of text and not buried in the figure legend of Fig. 1). Since Fanti et al. was comprehensively characterizing commercial HTT antibodies, I was very surprised that the commercial monoclonal HTT-MAB5490 and HTT-MAB2166 antibodies were used in their analyses. These two monoclonal HTT antibodies are widely used throughout the entire Huntington’s disease research community, and characterising these here (maybe instead of the MW1-MW8 series) would have been more appropriate / valuable. Methods: In the interest of having all the methodologies used in this already concise manuscript, it would be helpful to the reader to have all the experimental methodologies within this section, as opposed to viewing them from another link online. 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? Partly Are the datasets clearly presented in a useable and accessible format? Yes References 1. Ko J, Ou S, Patterson PH: New anti-huntingtin monoclonal antibodies: implications for huntingtin conformation and its binding proteins. Brain Res Bull . 56 (3-4): 319-29 PubMed Abstract | Publisher Full Text 2. Landles C, Sathasivam K, Weiss A, Woodman B, et al.: Proteolysis of mutant huntingtin produces an exon 1 fragment that accumulates as an aggregated protein in neuronal nuclei in Huntington disease. J Biol Chem . 2010; 285 (12): 8808-23 PubMed Abstract | Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise The molecular pathogenesis of Huntingtin's disease, pre-clinical Huntington's disease mouse models, HTT protein bioassays, Molecular biology, HTT protein aggregation. 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) Landles C. Peer Review Report For: A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.176211.r360584) 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/13-922/v2#referee-response-360584 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 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. 09 Sep 2024 | for Version 1 Karen Bowman , University of Leicester, Leicester, England, UK 0 Views copyright © 2024 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 article pointed out the current technical difficulties in studying Huntington’s Disease and identified the differences in antibodies to the protein Huntingtin, used throughout the HD research community, as a particular problem. The study formed part of the wider YCharOS collaboration to characterise commercial antibodies for human proteins using standardized protocols with the intention of improving antibody reproducibility issues. The study was an evaluation of twenty commercial antibodies for the protein Huntingtin for use in western blot, immunoprecipitation, and immunofluorescence. The study was well designed and the results were to a high technical standard and quality. The data is presented clearly and looks robust. It was reassuring to see that a committee of industry and academic representatives had endorsed the platform used. The platform consisted of identification of human cell lines suitable for antibody characterisation studies i.e. with adequate levels of Huntingtin, followed by the development of and contribution of isogenic knockout control cell lines, which were validated at the protein level. The final step was a series of antibody characterisation procedures, limited to the most common research uses of antibodies. Limitations of the study are clearly stated. Two minor errors were spotted. There is an inconsistency between the text and figure 3, where they mention IP was done on twenty antibodies in the text, but results for only ten antibodies are presented in the figure. Perhaps some additional text on why only ten were presented would be of use, or amend the text to reflect what is presented in the figure. A second error is that in the final results paragraph on immunofluorescence, figure 3 is cited, and linked back to, instead of figure 4. Overall, this will be a highly useful resource for scientists in the HD research community. 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 drug discovery and diagnostics 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 Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.168592.r315013) 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/13-922/v1#referee-response-315013 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 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. 28 Aug 2024 | for Version 1 Jieya Shao , Washington University in St Louis, St. Louis, Missouri, USA 0 Views copyright © 2024 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 study, the authors performed comprehensive analysis of twenty commercially available HTT antibodies for their suitability and performance in Western blot, immunoprecipitation, and immunofluorescence staining applications. The experimental approaches were logically and thoroughly designed. Data are of high-quality and clearly presented. The results will be very helpful for researchers in the HTT field. However, there seems to be two small errors. First, in Figure 3, only the IP data of 10 antibodies were shown while in the text it was stated that 20 antibodies were analyzed. Second, on Page 5, in the last sentence of the second paragraph, the authors seem to cite Figure 3 by mistake. 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) Shao J. Peer Review Report For: A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 5 approved, 1 approved with reservations] . F1000Research 2025, 13 :922 ( https://doi.org/10.5256/f1000research.168592.r315014) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. 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