A guide to selecting high-performing antibodies for Stearoyl-CoA desaturase (SCD1) (UniProt ID: O00767) for use in western blot, immunoprecipitation, and immunofluorescence

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Understanding the specific mechanisms by which SCD1 plays in health and disease can provide novel insides in therapeutic targets, a process that would be facilitated by the availability of high-quality antibodies. Here we have characterized nine SCD1 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/14-10", "name": "A guide to selecting high-performing antibodies for Stearoyl-CoA desaturase..." } } ] } Home Browse A guide to selecting high-performing antibodies for Stearoyl-CoA desaturase... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Ruíz Moleón V, Alende C, Fotouhi M et al. A guide to selecting high-performing antibodies for Stearoyl-CoA desaturase (SCD1) (UniProt ID: O00767) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :10 ( https://doi.org/10.12688/f1000research.160217.2 ) 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 Stearoyl-CoA desaturase (SCD1) (UniProt ID: O00767) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 1 approved, 2 approved with reservations] Vera Ruíz Moleón https://orcid.org/0000-0003-3728-3158 1 , Charles Alende https://orcid.org/0009-0005-4611-6134 1 , Maryam Fotouhi 1 , [...] Sara González Bolívar https://orcid.org/0000-0002-4299-8281 1 , Riham Ayoubi 1 , Carl Laflamme https://orcid.org/0000-0001-5906-025X 1 , NeuroSGC/YCharOS/EDDU collaborative group , ABIF consortium Vera Ruíz Moleón https://orcid.org/0000-0003-3728-3158 1 , Charles Alende https://orcid.org/0009-0005-4611-6134 1 , [...] Maryam Fotouhi 1 , Sara González Bolívar https://orcid.org/0000-0002-4299-8281 1 , Riham Ayoubi 1 , Carl Laflamme https://orcid.org/0000-0001-5906-025X 1 , NeuroSGC/YCharOS/EDDU collaborative group , ABIF consortium PUBLISHED 13 Jun 2025 Author details Author details 1 Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, Montreal, Québec, Canada Vera Ruíz Moleón Roles: Investigation Charles Alende Roles: Investigation Maryam Fotouhi Roles: Investigation Sara González Bolívar Roles: Investigation Riham Ayoubi Roles: Investigation, Supervision Carl Laflamme Roles: Conceptualization, Funding Acquisition, Project Administration OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the YCharOS (Antibody Characterization through Open Science) gateway. Abstract The enzyme stearoyl-CoA desaturase (SCD1) is a modulator of lipid metabolism by catalyzing the biosynthesis of mono-unsaturated fatty acids from saturated fatty acids. Understanding the specific mechanisms by which SCD1 plays in health and disease can provide novel insides in therapeutic targets, a process that would be facilitated by the availability of high-quality antibodies. Here we have characterized nine SCD1 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 O00767, SCD, SCD1, Steroyl-CoA desaturase, antibody characterization, antibody validation, western blot, immunoprecipitation, immunofluorescence Corresponding Author(s) Carl Laflamme ( [email protected] ) Close Corresponding author: Carl Laflamme Competing interests: For this project, the authors developed partnerships with high-quality antibody manufacturers and KO cell line providers. The partners provide antibodies and KO cell lines to the McPherson laboratory at no cost. These partners include: - Abcam- ABCD antibodies- ABclonal- Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific. Grant information: This work was supported by a grant from the Michael J. Fox Parkinson’s Disease Research. It was also supported by the Government of Canada through Genome Canada, Genome Quebec, and Ontario Genomics (grant no. OGI-210). RA is supported by a Mitacs fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 Ruíz Moleón V 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: Ruíz Moleón V, Alende C, Fotouhi M et al. A guide to selecting high-performing antibodies for Stearoyl-CoA desaturase (SCD1) (UniProt ID: O00767) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :10 ( https://doi.org/10.12688/f1000research.160217.2 ) First published: 02 Jan 2025, 14 :10 ( https://doi.org/10.12688/f1000research.160217.1 ) Latest published: 13 Jun 2025, 14 :10 ( https://doi.org/10.12688/f1000research.160217.2 ) Revised Amendments from Version 1 This revised version incorporates edits made in response to reviewer comments, which have improved the clarity of the manuscript. A new Table 3 has been added to assist antibody users in interpreting the data presented. Additionally, a limitations section has been included to acknowledge the constraints inherent to this antibody guide. This revised version incorporates edits made in response to reviewer comments, which have improved the clarity of the manuscript. A new Table 3 has been added to assist antibody users in interpreting the data presented. Additionally, a limitations section has been included to acknowledge the constraints inherent to this antibody guide. See the authors' detailed response to the review by Michael L. Garelja See the authors' detailed response to the review by Cecilia Williams and Matilda Holm READ REVIEWER RESPONSES Introduction Stearoyl-CoA desaturase (SCD1) is a membrane-bound enzyme which catalyzes the rate-limiting step in the conversion of saturated fatty acids into mono-unsaturated fatty acids. 1 , 2 The regulation of SCD1 is physiologically important as maintaining a proper ratio of saturated to monounsaturated fatty acids is essential for membrane fluidity. Disruption to this ratio can lead to pathological conditions, including cardiovascular disease, obesity, non-insulin dependent diabetes mellitus, hypertension, neurological diseases, immune disorders and cancer. 2 – 7 SCD1 is of particular importance in Parkinson’s disease (PD) research as its inhibition has been found to rescue α-Synuclein cytotoxicity and inclusion formation, both hallmarks of PD progression. The neurotoxic mechanisms underlying PD progression are not yet clearly defined. 8 – 10 Mechanistic studies would be facilitated with the availability of high-quality SCD1 antibodies. 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, 11 and openly sharing the data. 12 – 14 Here we evaluated the performance of nine commercial antibodies for SCD1 for use in western blot, immunoprecipitation, and immunofluorescence (also referred to as immunocytochemistry), enabling biochemical and cellular assessment of SCD1 properties and function. The platform for antibody characterization used to carry out this study was endorsed by a committee of industry academic representatives. It consists of identifying human cell lines with adequate target protein expression and the development/contribution of equivalent knockout (KO) cell lines, followed by antibody characterization procedures using most commercially available renewable antibodies against the corresponding protein. The standardized consensus antibody characterization protocols are openly available on Protocol Exchange (DOI: 10.21203/rs.3.pex-2607/v1 ). 15 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 16 and in Table 3 of this data note. Results and discussion Our standard protocol involves comparing readouts from WT (wild type) and KO cells. 17 , 18 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 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). The HeLa cell line expresses the SCD1 transcript at 6.7 log 2 (TPM+1) RNA levels, which is above the average range of cancer cells analyzed, and does not carry mutations in the SCD gene that could affect antibody–epitope binding, as seen on DepMap. A SCD KO HeLa cells were obtained from Abcam ( Table 1 ). Table 1. Summary of the cell lines used. Institution Catalog number RRID (Cellosaurus) Cell line Genotype Abcam ab255448 CVCL_0030 HeLa WT Abcam ab265220 CVCL_B2EP HeLa SCD KO 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 SCD1 antibodies. YCharOS partners provide approximately 80% of all renewable antibodies, but some top-cited polyclonal antibodies may not be available through these partners. Secondly, the YCharOS effort employs a non-biased approach that is agnostic to the protein for which antibodies have been characterized. The aim is to provide objective data on antibody performance without preconceived notions about how antibodies should perform or the molecular weight that should be observed in western blot. As the authors are not experts in SCD1, only a brief overview of the protein’s function and its relevance in disease is provided. SCD1 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. Moreover, the same antibody clones are often donated by 2–3 manufacturers—such as the SCD1 antibodies ab19862 and MA5-27542 (clone CD.E10, cross-licensed between Abcam and Thermo Fisher)—effectively serving as replicates and enabling validation of test reproducibility. 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. For western blot experiments, WT and SCD KO protein lysates were ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with nine antibodies in parallel ( Table 2 , Figure 1 ). Table 2. Summary of the SCD1 antibodies tested. Company Catalog number Lot number RRID (Antibody Registry) Clonality Clone ID Host Concentration (μg/μl) Vendors recommended applications Abcam ab19862 * 1057200-1 AB_445179 monoclonal CD.E10 mouse 1.00 Wb, IP, IF Abcam ab236868 ** 1007366-14 AB_2928123 recombinant mono EPR21963 rabbit 0.61 Wb, IP, IF Abcam ab39969 1036585-6 AB_945374 polyclonal rabbit 0.90 Wb Aviva Systems Biology ARP32797_T100 QC2226-43641 AB_841676 polyclonal rabbit 1.00 Wb Bio-Techne AF7550 CGOP0121061 AB_3107036 polyclonal sheep 0.20 Wb Proteintech 28678-1-AP 00103543 AB_2923581 polyclonal rabbit 0.40 Wb, IF Thermo Fisher Scientific MA5-27542 * YH4004441A AB_2723611 monoclonal CD.E10 mouse 1.00 Wb, IP, IF Thermo Fisher Scientific PA5-75757 YJ4089139 AB_2719485 polyclonal rabbit 1.00 Wb, IF Thermo Fisher Scientific PA5-95762 YJ4090059A AB_2807564 polyclonal rabbit 1.35 Wb, IF * Monoclonal antibody. ** Recombinant antibody. Figure 1. SCD1 antibody screening by western blot. Lysates of HeLa WT and SCD KO were prepared, and 35 μg of protein were processed for western blot with the indicated SCD1 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Tris-Glycine 4-20% gels were used. Antibody dilutions were chosen according to the recommendations of the antibody supplier. An exception was given for antibody AF7550 which was titrated because the signal was too weak when following the supplier’s recommendations. Antibody dilution used: ab19862* at 1/1000, ab236868** at 1/1000, ab39969 at 1/1000, ARP32797_T100 at 1/1000, AF7550 at 1/200, 28678-1-AP at 1/1000, MA5-27542* at 1/1000, PA5-75757 at 1/200 and PA5-95762 at 1/1000. Predicted band size: 41.5 kDa *Monoclonal antibody, **Recombinant antibody. We then assessed the capability of all nine antibodies to capture SCD1 from HeLa protein extracts using immunoprecipitation techniques, followed by western blot analysis. For the immunoblot step, a specific SCD1 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 2 ). Figure 2. SCD1 antibody screening by immunoprecipitation. HeLa lysates were prepared, and immunoprecipitation was performed using 1 mg of lysate and 2.0 μg of the indicated SCD1 antibodies pre-coupled to Dynabeads protein A or protein G. Samples were washed and processed for western blot with the indicated SCD1 antibody. For western blot, MA5-27542* was used at 1/1000. Tris-Glycine 4-20% gels were used. The Ponceau stained transfers of each blot are shown. Predicted band size: 41.5 kDa. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate, HC= antibody heavy chain, LC= antibody light chain. *Monoclonal antibody, **Recombinant antibody. For immunofluorescence, nine antibodies were screened using a mosaic strategy. First, HeLa WT and SCD KO cells were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the SCD1 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. 15 Figure 3. SCD1 antibody screening by immunofluorescence. HeLa WT and SCD 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 SCD1 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: ab19862* at 1/1000, ab236868** at 1/600, ab39969 at 1/150, ARP32797_T100 at 1/500, AF7550 at 1/200, 28678-1-AP at 1/400, MA5-27542* at 1/1000, PA5-75757 at 1/1000 and PA5-95762 at 1/1300. Bars = 10 μm. *Monoclonal antibody, **Recombinant antibody. Table 3. Illustrations to assess antibody performance in all western blot, immunoprecipitation and immunofluorescence. Western blot Immunoprecipitation Immunofluorescence In conclusion, we have screened nine SCD1 commercial antibodies by western blot, immunoprecipitation, and immunofluorescence by comparing the signal produced by the antibodies in human HeLa WT and SCD KO cells. High-quality and renewable antibodies that successfully detect SCD1 were identified in all applications. Researchers who wish to study SCD1 in a different species are encouraged to select high-quality antibodies based on the results presented and investigate the predicted species reactivity of the manufacturer before extending their research. Method The standardized protocols used to carry out this KO cell line-based antibody characterization platform was established and approved by a collaborative group of academics, industry researchers and antibody manufacturers. The detailed materials and step-by-step protocols used to characterize antibodies in western blot, immunoprecipitation and immunofluorescence are openly available on Protocol Exchange (DOI: 10.21203/rs.3.pex-2607/v1 ). 15 Antibodies and cell line 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. 19 , 20 All cell lines used in this study were regularly tested for mycoplasma contamination and were confirmed to be mycoplasma-free. Data availability Underlying data Zenodo: Antibody Characterization Report for SCD1, https://doi.org/10.5281/zenodo.13891494 . 21 Zenodo: Dataset for the SCD1 antibody screening study, https://doi.org/10.5281/zenodo.14502183 . 22 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. Enoch HG, Catalá A, Strittmatter P: Mechanism of rat liver microsomal stearyl-CoA desaturase. Studies of the substrate specificity, enzyme-substrate interactions, and the function of lipid. J. Biol. Chem. 1976; 251 (16): 5095–5103. PubMed Abstract | Publisher Full Text 2. Ntambi JM: Regulation of stearoyl-CoA desaturase by polyunsaturated fatty acids and cholesterol. J. Lipid Res. 1999; 40 (9): 1549–1558. Publisher Full Text 3. Kinsella JE, Lokesh B, Stone RA: Dietary n-3 polyunsaturated fatty acids and amelioration of cardiovascular disease: possible mechanisms. Am. J. Clin. Nutr. 1990; 52 (1): 1–28. PubMed Abstract | Publisher Full Text 4. Jones BH, Maher MA, Banz WJ, et al. : Adipose tissue stearoyl-CoA desaturase mRNA is increased by obesity and decreased by polyunsaturated fatty acids. Am. J. Phys. 1996; 271 (1 Pt 1): E44–E49. Publisher Full Text 5. Li J, Ding SF, Habib NA, et al. : Partial characterization of a cDNA for human stearoyl-CoA desaturase and changes in its mRNA expression in some normal and malignant tissues. Int. J. Cancer. 1994; 57 (3): 348–352. PubMed Abstract | Publisher Full Text 6. Habib NA, Wood CB, Apostolov K, et al. : Stearic acid and carcinogenesis. Br. J. Cancer. 1987; 56 (4): 455–458. PubMed Abstract | Publisher Full Text | Free Full Text 7. Khoo DE, Fermor B, Miller J, et al. : Manipulation of body fat composition with sterculic acid can inhibit mammary carcinomas in vivo. Br. J. Cancer. 1991; 63 (1): 97–101. PubMed Abstract | Publisher Full Text | Free Full Text 8. Fanning S, Haque A, Imberdis T, et al. : Lipidomic Analysis of α-Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment. Mol. Cell. 2019; 73 (5): 1001–1014.e8. PubMed Abstract | Publisher Full Text | Free Full Text 9. Bartels T, Choi JG, Selkoe DJ: α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation. Nature. 2011; 477 (7362): 107–110. PubMed Abstract | Publisher Full Text | Free Full Text 10. Nicholatos JW, Groot J, Dhokai S, et al. : SCD Inhibition Protects from α-Synuclein-Induced Neurotoxicity But Is Toxic to Early Neuron Cultures. eNeuro. 2021; 8 (4): ENEURO.0166–ENEU21.2021. PubMed Abstract | Publisher Full Text | Free Full Text 11. Ayoubi R, Ryan J, Gonzalez Bolivar S, et al. : A consensus platform for antibody characterization. Nat. Protoc. 2025 Jun; 20 (6): 1509–1545. PubMed Abstract | Publisher Full Text 12. 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 13. 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 14. 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 15. Ayoubi R, Ryan J, Bolivar SG, et al. : A consensus platform for antibody characterization (Version 1). Protocol Exchange. 2024. 16. Biddle MS, Virk HS: YCharOS open antibody characterisation data: Lessons learned and progress made. F1000Res. 2023; 12 : 1344. Publisher Full Text 17. 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 18. 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 19. 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 20. 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 21. Ruiz Moleon V, Alende C, Fotouhi M, et al. : A guide to selecting high-performing antibodies for Stearoyl-CoA desaturase (SCD1) (UniProt ID: O00767). Zenodo. 2024. Publisher Full Text 22. Laflamme C: Dataset for the SCD1 antibody screening study. [Dataset]. Zenodo. 2024. Publisher Full Text Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 02 Jan 2025 ADD YOUR COMMENT Comment Author details Author details 1 Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, Montreal, Québec, Canada Vera Ruíz Moleón Roles: Investigation Charles Alende Roles: Investigation Maryam Fotouhi Roles: Investigation Sara González Bolívar Roles: Investigation Riham Ayoubi Roles: Investigation, Supervision Carl Laflamme Roles: Conceptualization, Funding Acquisition, Project Administration Competing interests For this project, the authors developed partnerships with high-quality antibody manufacturers and KO cell line providers. The partners provide antibodies and KO cell lines to the McPherson laboratory at no cost. These partners include: - Abcam- ABCD antibodies- ABclonal- Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific. Grant information This work was supported by a grant from the Michael J. Fox Parkinson’s Disease Research. It was also supported by the Government of Canada through Genome Canada, Genome Quebec, and Ontario Genomics (grant no. OGI-210). RA is supported by a Mitacs fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (2) version 2 Revised Published: 13 Jun 2025, 14:10 https://doi.org/10.12688/f1000research.160217.2 version 1 Published: 02 Jan 2025, 14:10 https://doi.org/10.12688/f1000research.160217.1 Copyright © 2025 Ruíz Moleón V 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 Ruíz Moleón V, Alende C, Fotouhi M et al. A guide to selecting high-performing antibodies for Stearoyl-CoA desaturase (SCD1) (UniProt ID: O00767) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :10 ( https://doi.org/10.12688/f1000research.160217.2 ) 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 13 Jun 2025 Revised Views 0 Cite How to cite this report: Moshinsky D. Reviewer Report For: A guide to selecting high-performing antibodies for Stearoyl-CoA desaturase (SCD1) (UniProt ID: O00767) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :10 ( https://doi.org/10.5256/f1000research.183557.r392564 ) The direct URL for this report is: https://f1000research.com/articles/14-10/v2#referee-response-392564 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 10 Jul 2025 Deborah Moshinsky , Institute for Protein Innovation, Boston, Massachusetts, USA Approved VIEWS 0 https://doi.org/10.5256/f1000research.183557.r392564 The authors tested 9 commercially available antibodies to SCD1 (stearoyl-CoA desaturase) for activity in Western Blot, Immunoprecipitation (IP) and immunofluorescence (IF). They first found a cell line expressing the protein and generated a knockout version. Then they utilized standardized, openly ... Continue reading READ ALL The authors tested 9 commercially available antibodies to SCD1 (stearoyl-CoA desaturase) for activity in Western Blot, Immunoprecipitation (IP) and immunofluorescence (IF). They first found a cell line expressing the protein and generated a knockout version. Then they utilized standardized, openly available protocols for Western, IP and IF. The results were clear and the methodology easy to follow. Table 3 is useful to assist in the interpretation of the data. However, for the Western Blot examples in Table 3, the situation where multiple bands including the target are present in the WT and multiple bands but lacking the target is present in the KO is labeled as a 'successful antibody.' I recommend that this be clarified, as one could argue that an antibody that detects it's target along with other proteins is non-specific and therefore not a 'successful' antibody. Perhaps successful but non-selective or another qualifying term would be more suitable. Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Antibody 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 Stearoyl-CoA desaturase (SCD1) (UniProt ID: O00767) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :10 ( https://doi.org/10.5256/f1000research.183557.r392564 ) The direct URL for this report is: https://f1000research.com/articles/14-10/v2#referee-response-392564 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 02 Jan 2025 Views 0 Cite How to cite this report: Garelja ML. Reviewer Report For: A guide to selecting high-performing antibodies for Stearoyl-CoA desaturase (SCD1) (UniProt ID: O00767) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :10 ( https://doi.org/10.5256/f1000research.176074.r358436 ) The direct URL for this report is: https://f1000research.com/articles/14-10/v1#referee-response-358436 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 Michael L. Garelja , University of Otago, Dunedin, New Zealand Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.176074.r358436 This manuscript, aiming to investigate the utility of a range of SCD1 antibodies is extremely important, and I applaud the initiative. Identification of antibodies that can correctly detect their target is of incredible importance to the entire field and highlighting ... Continue reading READ ALL This manuscript, aiming to investigate the utility of a range of SCD1 antibodies is extremely important, and I applaud the initiative. Identification of antibodies that can correctly detect their target is of incredible importance to the entire field and highlighting that not all antibodies work as advertised is critical to ensuring that researchers can perform robust science. Points that need be addressed for this work to be indexed. Are there RRID’s associated with these antibodies? This applies for both the primary antibodies, and the secondary antibodies. Secondary antibodies also require lot numbers. As antibody concentrations can change between batches, it would be more informative to list the concentrations used in the experiments rather than the dilutions. A summary table to show how the different antibodies work under different experimental techniques would be extremely useful. I understand that the resource cannot make recommendations, but having a summary of how each antibody would help others interpret results. Are there any known splice variants of this target? There is a consistent band of approximately 25 kDa, that also seems to be lower when knocked out. Any insight as to why the detected band differed from the estimated molecular weight would also be useful. Has this particular ladder been compared to other ladders to ensure that it runs as expected under these experimental conditions? Further information for discussion. The researchers have used a cell-line which highly expresses the target. Is it possible to work with cells that have lower expression levels? If not, these needs to be addressed as a limitation. Testing the panel of “useful” antibodies against more cell lines with variable SCD1 expression to show a limit of detection would be of high utility. Listing the epitopes that the antibodies were raised against would be of high value. One of the pillars of antibody validation is to use two antibodies that have been raised against different epitopes, so having that information readily available would help researchers in their mission to perform good science. The paper should acknowledge that there may be methodological optimizations that could improve antibody function. Figure 3 – is it possible to show to green and red stained cells in a separate panel? The outlines are useful, but showing that the cells have similar morphologies (or not!) would be interesting in the context of SCD1 biology. Why were these antibodies chosen, as opposed to the wider range of antibodies available? This is not a criticism, as the work is valuable regardless, but some information as to how these antibodies were selected would be interesting. 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 Competing Interests: No competing interests were disclosed. Reviewer Expertise: CGRP, migraine, pharmacology 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 Garelja ML. Reviewer Report For: A guide to selecting high-performing antibodies for Stearoyl-CoA desaturase (SCD1) (UniProt ID: O00767) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :10 ( https://doi.org/10.5256/f1000research.176074.r358436 ) The direct URL for this report is: https://f1000research.com/articles/14-10/v1#referee-response-358436 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 10 Sep 2025 Carl Laflamme , Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, Montreal, Canada 10 Sep 2025 Author Response Dear Dr. Garelja, Thank you for your encouraging words and thoughtful suggestions. We greatly appreciate your support of the YCharOS initiative and your detailed feedback, which helped us further ... Continue reading Dear Dr. Garelja, Thank you for your encouraging words and thoughtful suggestions. We greatly appreciate your support of the YCharOS initiative and your detailed feedback, which helped us further strengthen the manuscript. Please find our responses to your comments below: 1. RRIDs for antibodies: We have carefully assigned RRIDs to all primary antibodies. In this report, polyclonal secondary antibodies were used. However, we are transitioning toward the exclusive use of recombinant secondary antibodies, and RRIDs will be included for those reagents in future studies. 2. Reporting concentrations vs. dilutions: We understand the rationale for suggesting antibody concentrations. However, to maintain consistency with manufacturer guidelines and accommodate proprietary formulations (where concentrations are not disclosed), we report antibody usage as dilutions, as recommended by most vendors. 3. Summary of antibody performance: We have added a new Table (Table 3) to provide a schematic summary of antibody performance across applications. While we do not rank antibodies, this overview will help users interpret the data more efficiently. 4. Unexpected SDS-PAGE migration / lower molecular weight band: As this is an agnostic antibody validation effort, we refrain from interpreting specific bands. Nonetheless, we agree this is a valuable point for discussion. Proteins may migrate differently than expected on SDS-PAGE due to post-translational modifications, unusual amino acid composition, protein folding, or incomplete denaturation, which can affect SDS binding and alter migration behavior. The consistent ~25 kDa band and its reduction in the knockout sample may represent a splice variant, degradation product, or non-specific interaction. We have included this discussion in the revised manuscript. Minor Comments 1. Use of a high-expressing cell line: We agree this is a limitation. SCD1 is broadly expressed and considered an essential gene. HeLa cells express SCD1 slightly below the median level across cancer cell lines (6.7 vs. 7.0 TPM; DepMap data). We now highlight this rationale and limitation explicitly in the revised manuscript. 2. Epitope: As a public initiative, our focus is to provide KO-based, standardized antibody validation across key applications. While we value reviewer and community suggestions, incorporating additional information would introduce burdens that could limit the scalability and efficiency of the initiative. 3. Methodological optimization: We have acknowledged in the revised text that further optimization of protocols may improve antibody performance and encourage researchers to adapt conditions to their specific needs. 4. Raw data accessibility: All raw data files are openly available via Zenodo, as described in the Data Availability section of the manuscript. 5. Antibody selection criteria: The antibodies included in this study were provided free of charge by participating manufacturers. Selection was based on availability, vendor prioritization, and a preference for renewable reagents where possible. We have clarified this in the revised manuscript. Thank you for your comments, which have helped us improve the clarity and rigor of this antibody characterization guide. Sincerely, Carl Laflamme (on behalf of all co-authors) Dear Dr. Garelja, Thank you for your encouraging words and thoughtful suggestions. We greatly appreciate your support of the YCharOS initiative and your detailed feedback, which helped us further strengthen the manuscript. Please find our responses to your comments below: 1. RRIDs for antibodies: We have carefully assigned RRIDs to all primary antibodies. In this report, polyclonal secondary antibodies were used. However, we are transitioning toward the exclusive use of recombinant secondary antibodies, and RRIDs will be included for those reagents in future studies. 2. Reporting concentrations vs. dilutions: We understand the rationale for suggesting antibody concentrations. However, to maintain consistency with manufacturer guidelines and accommodate proprietary formulations (where concentrations are not disclosed), we report antibody usage as dilutions, as recommended by most vendors. 3. Summary of antibody performance: We have added a new Table (Table 3) to provide a schematic summary of antibody performance across applications. While we do not rank antibodies, this overview will help users interpret the data more efficiently. 4. Unexpected SDS-PAGE migration / lower molecular weight band: As this is an agnostic antibody validation effort, we refrain from interpreting specific bands. Nonetheless, we agree this is a valuable point for discussion. Proteins may migrate differently than expected on SDS-PAGE due to post-translational modifications, unusual amino acid composition, protein folding, or incomplete denaturation, which can affect SDS binding and alter migration behavior. The consistent ~25 kDa band and its reduction in the knockout sample may represent a splice variant, degradation product, or non-specific interaction. We have included this discussion in the revised manuscript. Minor Comments 1. Use of a high-expressing cell line: We agree this is a limitation. SCD1 is broadly expressed and considered an essential gene. HeLa cells express SCD1 slightly below the median level across cancer cell lines (6.7 vs. 7.0 TPM; DepMap data). We now highlight this rationale and limitation explicitly in the revised manuscript. 2. Epitope: As a public initiative, our focus is to provide KO-based, standardized antibody validation across key applications. While we value reviewer and community suggestions, incorporating additional information would introduce burdens that could limit the scalability and efficiency of the initiative. 3. Methodological optimization: We have acknowledged in the revised text that further optimization of protocols may improve antibody performance and encourage researchers to adapt conditions to their specific needs. 4. Raw data accessibility: All raw data files are openly available via Zenodo, as described in the Data Availability section of the manuscript. 5. Antibody selection criteria: The antibodies included in this study were provided free of charge by participating manufacturers. Selection was based on availability, vendor prioritization, and a preference for renewable reagents where possible. We have clarified this in the revised manuscript. Thank you for your comments, which have helped us improve the clarity and rigor of this antibody characterization guide. Sincerely, Carl Laflamme (on behalf of all co-authors) Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 10 Sep 2025 Carl Laflamme , Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, Montreal, Canada 10 Sep 2025 Author Response Dear Dr. Garelja, Thank you for your encouraging words and thoughtful suggestions. We greatly appreciate your support of the YCharOS initiative and your detailed feedback, which helped us further ... Continue reading Dear Dr. Garelja, Thank you for your encouraging words and thoughtful suggestions. We greatly appreciate your support of the YCharOS initiative and your detailed feedback, which helped us further strengthen the manuscript. Please find our responses to your comments below: 1. RRIDs for antibodies: We have carefully assigned RRIDs to all primary antibodies. In this report, polyclonal secondary antibodies were used. However, we are transitioning toward the exclusive use of recombinant secondary antibodies, and RRIDs will be included for those reagents in future studies. 2. Reporting concentrations vs. dilutions: We understand the rationale for suggesting antibody concentrations. However, to maintain consistency with manufacturer guidelines and accommodate proprietary formulations (where concentrations are not disclosed), we report antibody usage as dilutions, as recommended by most vendors. 3. Summary of antibody performance: We have added a new Table (Table 3) to provide a schematic summary of antibody performance across applications. While we do not rank antibodies, this overview will help users interpret the data more efficiently. 4. Unexpected SDS-PAGE migration / lower molecular weight band: As this is an agnostic antibody validation effort, we refrain from interpreting specific bands. Nonetheless, we agree this is a valuable point for discussion. Proteins may migrate differently than expected on SDS-PAGE due to post-translational modifications, unusual amino acid composition, protein folding, or incomplete denaturation, which can affect SDS binding and alter migration behavior. The consistent ~25 kDa band and its reduction in the knockout sample may represent a splice variant, degradation product, or non-specific interaction. We have included this discussion in the revised manuscript. Minor Comments 1. Use of a high-expressing cell line: We agree this is a limitation. SCD1 is broadly expressed and considered an essential gene. HeLa cells express SCD1 slightly below the median level across cancer cell lines (6.7 vs. 7.0 TPM; DepMap data). We now highlight this rationale and limitation explicitly in the revised manuscript. 2. Epitope: As a public initiative, our focus is to provide KO-based, standardized antibody validation across key applications. While we value reviewer and community suggestions, incorporating additional information would introduce burdens that could limit the scalability and efficiency of the initiative. 3. Methodological optimization: We have acknowledged in the revised text that further optimization of protocols may improve antibody performance and encourage researchers to adapt conditions to their specific needs. 4. Raw data accessibility: All raw data files are openly available via Zenodo, as described in the Data Availability section of the manuscript. 5. Antibody selection criteria: The antibodies included in this study were provided free of charge by participating manufacturers. Selection was based on availability, vendor prioritization, and a preference for renewable reagents where possible. We have clarified this in the revised manuscript. Thank you for your comments, which have helped us improve the clarity and rigor of this antibody characterization guide. Sincerely, Carl Laflamme (on behalf of all co-authors) Dear Dr. Garelja, Thank you for your encouraging words and thoughtful suggestions. We greatly appreciate your support of the YCharOS initiative and your detailed feedback, which helped us further strengthen the manuscript. Please find our responses to your comments below: 1. RRIDs for antibodies: We have carefully assigned RRIDs to all primary antibodies. In this report, polyclonal secondary antibodies were used. However, we are transitioning toward the exclusive use of recombinant secondary antibodies, and RRIDs will be included for those reagents in future studies. 2. Reporting concentrations vs. dilutions: We understand the rationale for suggesting antibody concentrations. However, to maintain consistency with manufacturer guidelines and accommodate proprietary formulations (where concentrations are not disclosed), we report antibody usage as dilutions, as recommended by most vendors. 3. Summary of antibody performance: We have added a new Table (Table 3) to provide a schematic summary of antibody performance across applications. While we do not rank antibodies, this overview will help users interpret the data more efficiently. 4. Unexpected SDS-PAGE migration / lower molecular weight band: As this is an agnostic antibody validation effort, we refrain from interpreting specific bands. Nonetheless, we agree this is a valuable point for discussion. Proteins may migrate differently than expected on SDS-PAGE due to post-translational modifications, unusual amino acid composition, protein folding, or incomplete denaturation, which can affect SDS binding and alter migration behavior. The consistent ~25 kDa band and its reduction in the knockout sample may represent a splice variant, degradation product, or non-specific interaction. We have included this discussion in the revised manuscript. Minor Comments 1. Use of a high-expressing cell line: We agree this is a limitation. SCD1 is broadly expressed and considered an essential gene. HeLa cells express SCD1 slightly below the median level across cancer cell lines (6.7 vs. 7.0 TPM; DepMap data). We now highlight this rationale and limitation explicitly in the revised manuscript. 2. Epitope: As a public initiative, our focus is to provide KO-based, standardized antibody validation across key applications. While we value reviewer and community suggestions, incorporating additional information would introduce burdens that could limit the scalability and efficiency of the initiative. 3. Methodological optimization: We have acknowledged in the revised text that further optimization of protocols may improve antibody performance and encourage researchers to adapt conditions to their specific needs. 4. Raw data accessibility: All raw data files are openly available via Zenodo, as described in the Data Availability section of the manuscript. 5. Antibody selection criteria: The antibodies included in this study were provided free of charge by participating manufacturers. Selection was based on availability, vendor prioritization, and a preference for renewable reagents where possible. We have clarified this in the revised manuscript. Thank you for your comments, which have helped us improve the clarity and rigor of this antibody characterization guide. Sincerely, Carl Laflamme (on behalf of all co-authors) Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Williams C and Holm M. Reviewer Report For: A guide to selecting high-performing antibodies for Stearoyl-CoA desaturase (SCD1) (UniProt ID: O00767) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :10 ( https://doi.org/10.5256/f1000research.176074.r355980 ) The direct URL for this report is: https://f1000research.com/articles/14-10/v1#referee-response-355980 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 17 Jan 2025 Cecilia Williams , KTH Royal Institute of Technology,, Stockholm, Sweden Matilda Holm , Protein Science, KTH Royal Institute of Technology (Ringgold ID: 7655), Stockholm, Stockholm County, Sweden Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.176074.r355980 The data note by Ruiz Moleon et al. provides a high-quality antibody validation guide for the protein SCD1. The guide is part of the YCharOS initiative that uses isogenic knockout cell lines and multiple antibody-dependent assays to investigate antibody specificity ... Continue reading READ ALL The data note by Ruiz Moleon et al. provides a high-quality antibody validation guide for the protein SCD1. The guide is part of the YCharOS initiative that uses isogenic knockout cell lines and multiple antibody-dependent assays to investigate antibody specificity and selectivity in a structured manner. It is an important effort that can improve the reproducibility of biomedical research, and this guide is highly useful for researchers focusing on SCD1. The authors show that 3-4 out of 9 antibodies do not work in most or any of the assays, whereas the rest are more or less well-performing, using the cells and conditions applied in the protocol. This is critical information that will be of high value to the research community. For enhanced usability, clarification, and stringency of the guide and to facilitate the interpretation and reproducibility, I have the below recommendations. Major comments The introduction would benefit from including basic information regarding the protein, as this would facilitate the interpretation of the data. Such as the known or predicted subcellular localization of SCD1, its calculated molecular weight, and if any splice variants are known. The rationale, as stated (p.3, 2 nd paragraph: “ Mechanistic studies would be facilitated with the availability of high-quality SCD1 antibodies ”) should be better supported. Is there a documented lack of high-quality SCD1 antibodies? Or, is this an assumption based on the general problem with the reproducibility of antibody-based research? The sentence “ using most commercially available antibodies against the corresponding protein” (p. 3, 3 rd paragraph) is likely not correct. Perhaps the authors intended to state “ renewable antibodies” as the initiative states in other publications. In this guide, specifically, the authors include 9 commercial (renewable and non-renewable) SCD1 antibodies, but there are as many as 274 antibodies from 32 providers directed towards human SCD1 listed in Antibodypedia. A statement of whether the SCD1 gene in the cells (HeLa WT) has indeed been confirmed to be wild type (i.e., not mutated) should be added. It would also be useful to include which (if any) splice variant transcripts are expressed in these cells. Table 1: The lot (or batch) numbers for the antibodies must be listed. This is essential information and is especially important in an antibody validation guide. It should be stated whether replicate experiments were performed. It is unclear how the antibody used for the western blot of the IPs was chosen (p. 3, 2 nd last paragraph: “ a specific antibody was chosen ”). Were both specificity and selectivity considered? Were the species the antibodies were generated in used as criteria (to avoid detection of heavy and light chains in the next step)? Or was the selection random out of the sufficiently specific antibodies? If so, how many and which antibodies were deemed to be sufficiently specific? Fig 2: Considering that antibodies from different species appear specific in WB, it seems like a missed opportunity to not take species into account to generate clearer western blot data of the IPs (i.e., avoiding additional bands of heavy and light antibody chains, such as when using mouse-generated antibodies in both IP and WB and detecting them with an anti-mouse secondary antibody). Fig. 1 and 2: It should be specified which secondary antibody was used in the western blots. Fig. 3: The legend statement that “ Representative images of the merged blue and red (grayscale) channels are shown. ” appears incorrect. The blue (DAPI) is shown separately from the red (antibody staining); they are not merged in the figure. It would also be clearer if the images showed the actual colors (blue for DAPI and red for SCD1 antibody) instead of grey for both. Only one cell line is used, which is a limitation of the initiative. Considering that HeLa cells express unusually high SCD1 levels (as stated in the text), the antibodies may work less well at lower SCD1 levels. They could also bind to other proteins expressed in other cells but not HeLa. It would be useful if the antibodies (the ones deemed to be of high quality) were additionally analyzed by western blot on a panel of cell lines with varying mRNA expression of SCD1 . This would indicate their performance at different levels of the target protein and in more than one cell line. Although the authors state that they do not engage in result analysis, they do (to select a specific antibody for WB of the IP, and they conclude that several antibodies successfully detect SCD1), but they refrain from clearly stating which these antibodies are. A table, or a statement, summarizing which antibodies bind intended and unintended targets in the different assays (under the specific conditions used) would allow greater usability, including for researchers starting their work (such as postdocs and PhD students). This would also facilitate an overview of the application-dependent performance (again, under the specific conditions used). Minor comments: The assay immunofluorescence (IF) is typically used for tissue analysis. For analysis of cells and cell lines, as is the case here, the term immunocytochemistry is better suited. UniProt ID may not need to be stated in the title (could be moved to abstract or introduction) p.3, 3 rd paragraph: It should be clarified if the target protein expression relates specifically to endogenous expression or whether cell lines with recombinant expression were included. A statement about mycoplasma testing and cell line authentication should be included. It would be beneficial to include which epitope each antibody is directed toward (or raised against). Fig. 1 and 2: The exposure time(s) applied for the western blots could be included. Fig 2: Although the figure legend clearly states which antibody was used for WB of all IPs, the figure itself gives the impression that it may have been used only for the two indicated IPs. It might be better to remove this from the image or place it differently. p. 5, first paragraph: “ Quantification of ... in hundreds of WT and KO cells ” is vague. The number (or range) of actually quantified cells should be stated. Fig. 3: The figure legend should specify which magnification was used for the IF images. It would be beneficial to highlight that the antibodies ab19862 and MA-27542 are from the same clone and should be the same. It would be pedagogical to place them side by side. Page 5, final paragraph: “ Several high-quality and renewable antibodies that successfully detect SCD1 were identified in all applications”. As only 3 of the tested antibodies were renewable (monoclonal or recombinant), and two of them were from the same clone (= same antibody), the word “several” seems a stretch. The word “renewable” should probably be removed. Several spelling/grammar errors (p.3 a “to” too much p.3, p. 3 “were run “ instead of “were ran”, wrongly placed commas, etc.) The published method protocol Ayoubi et al., Nature Protocol (2024) should likely be referenced. Author roles: It is not stated who drafted the manuscript, nor if all authors commented on it. 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? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: nuclear receptors, transcriptomes, antibody validation, cancer We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Williams C and Holm M. Reviewer Report For: A guide to selecting high-performing antibodies for Stearoyl-CoA desaturase (SCD1) (UniProt ID: O00767) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :10 ( https://doi.org/10.5256/f1000research.176074.r355980 ) The direct URL for this report is: https://f1000research.com/articles/14-10/v1#referee-response-355980 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 11 Sep 2025 Carl Laflamme , Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, Montreal, Canada 11 Sep 2025 Author Response Dear Dr. Williams and Dr. Holm, Thank you for your thoughtful review and constructive comments on our manuscript. We appreciate your recognition of the value and reproducibility aims of ... Continue reading Dear Dr. Williams and Dr. Holm, Thank you for your thoughtful review and constructive comments on our manuscript. We appreciate your recognition of the value and reproducibility aims of the YCharOS initiative, and we have revised the manuscript accordingly. Please find below our responses to your major and minor comments: Major comments: 1. Inclusion of basic protein information (subcellular localization, molecular weight, splice variants): We appreciate the suggestion. However, we deliberately avoid including expected localization or molecular weight data to maintain an agnostic and unbiased evaluation of antibody performance. Including such information can lead to confirmation bias, particularly when unreliable antibodies have historically led to incorrect assumptions about localization or molecular weight. While the theoretical molecular weight of SCD1 is provided in the Figure 1 legend, we acknowledge that SDS-PAGE migration can deviate due to post-translational modifications or proteolytic processing. 2. Rationale for selecting SCD1 as a target: The study was funded by the Simons Foundation Autism Research Initiative (SFARI), which surveyed its community to identify autism-relevant proteins lacking high-quality research tools. SCD1 was among seven prioritized targets identified for systematic antibody validation. This supports the relevance and urgency of the work. 3. Use of the term “most commercially available antibodies”: We agree with the reviewer. The sentence has been revised to refer specifically to “renewable antibodies,” which aligns with the language used in our previous publications. We also note that polyclonal antibodies are often cross-licensed under different catalog numbers, complicating estimates of the actual number of unique reagents. 4. Confirmation of HeLa WT status and splice variants: According to DepMap.org, the SCD1 gene is not mutated in HeLa cells. We have added this information to the manuscript. Consistent with our agnostic approach (see point 1), we have chosen not to include specific splice variant information, as we cannot guarantee their detectability under the conditions used. 5. Lot numbers for antibodies: Thank you for pointing this out. The lot numbers were already included in Table 1 (third column). 6. Replicates and reproducibility: We have added a new “Limitations” section preceding the Methods to address this point more explicitly. In brief, replicates are addressed indirectly through use of cross-licensed antibodies from different manufacturers. Experiments are standardized using master mixes and consistent protocols. Where signal is absent, repeat experiments are conducted. For IF, dual concentrations are tested to assess performance. 7–9. Choice of antibody for WB of IPs and secondary antibodies used: We have revised the manuscript to clarify that MA5-27542 (a mouse monoclonal) was selected for immunoblotting of immunoprecipitates to avoid species overlap, as most tested IP antibodies are rabbit-derived. Using the same antibody across all IP samples facilitates direct performance comparison. The correct secondary antibodies were used in all applications, and their identities have been specified in the revised manuscript. 10. IF image display and labeling: Thank you for noting this. The legend now correctly indicates that separate (not merged) grayscale images are shown. We have retained grayscale display, consistent with microscopy best practices for visual clarity. 11. Use of a single cell line: We agree that this is a limitation. SCD1 is broadly expressed and considered an essential gene. HeLa cells express SCD1 at a level slightly below the median across cancer cell lines analyzed by DepMap (6.7 vs. 7.0 TPM). We now highlight this limitation and rationale in the revised manuscript. 12. Clarification of high-quality antibodies and application-specific summary: While we avoid drawing formal conclusions on antibody performance, we do indicate those used in key figures (e.g., Figures 2 and 3). As noted, interpretation is left to the end user, with guidance provided in the editorial accompanying our gateway (DOI: 10.12688/f1000research.141719.1). We also added Table 3 that guides researchers interpret validation data. Minor comments: Terminology (IF vs ICC): The manuscript now includes both terms for clarity. UniProt ID in title: We retained the UniProt ID in the title due to the high variability in protein names, which helps ensure accurate identification. Clarification of endogenous expression: The relevant section has been revised to clarify that all expression pertains to endogenous protein levels. Mycoplasma testing and cell line authentication: A statement regarding mycoplasma testing and cell line authentication has been added to the Methods section. Epitope information: As a public initiative, our focus is to provide KO-based, standardized antibody validation across key applications. While we value reviewer and community suggestions, incorporating additional information would introduce burdens that could limit the scalability and efficiency of the initiative. Exposure times for WB: We have chosen not to include exposure times, as they vary with protein loading, detection systems, and antibody dilutions, which would limit reproducibility across labs. Antibody label in IP figure: We have decided to keep the labelling as it is. Cell quantification in IF: We now specify that at least 215 wild-type and knockout cells were quantified, addressing the reviewer’s concern. Magnification in IF figures: The magnification is specified in the figure legend (scale bar = 10 µm). Related antibodies (clones): We now explicitly state that ab19862 and MA5-27542 originate from the same clone. Clarification of renewable antibodies: We agree and have revised the statement in question. Since only two unique renewable antibodies were tested, we removed the word “several.” Grammar and spelling corrections: All grammar, spelling, and punctuation issues flagged have been addressed in the revised manuscript. Citation of protocol paper: Ayoubi et al., Nature Protocols (2024) has been properly cited. We are grateful for your insightful comments, which have helped us improve the clarity and rigor of this antibody characterization guide. Sincerely, Carl Laflamme (on behalf of all co-authors) Dear Dr. Williams and Dr. Holm, Thank you for your thoughtful review and constructive comments on our manuscript. We appreciate your recognition of the value and reproducibility aims of the YCharOS initiative, and we have revised the manuscript accordingly. Please find below our responses to your major and minor comments: Major comments: 1. Inclusion of basic protein information (subcellular localization, molecular weight, splice variants): We appreciate the suggestion. However, we deliberately avoid including expected localization or molecular weight data to maintain an agnostic and unbiased evaluation of antibody performance. Including such information can lead to confirmation bias, particularly when unreliable antibodies have historically led to incorrect assumptions about localization or molecular weight. While the theoretical molecular weight of SCD1 is provided in the Figure 1 legend, we acknowledge that SDS-PAGE migration can deviate due to post-translational modifications or proteolytic processing. 2. Rationale for selecting SCD1 as a target: The study was funded by the Simons Foundation Autism Research Initiative (SFARI), which surveyed its community to identify autism-relevant proteins lacking high-quality research tools. SCD1 was among seven prioritized targets identified for systematic antibody validation. This supports the relevance and urgency of the work. 3. Use of the term “most commercially available antibodies”: We agree with the reviewer. The sentence has been revised to refer specifically to “renewable antibodies,” which aligns with the language used in our previous publications. We also note that polyclonal antibodies are often cross-licensed under different catalog numbers, complicating estimates of the actual number of unique reagents. 4. Confirmation of HeLa WT status and splice variants: According to DepMap.org, the SCD1 gene is not mutated in HeLa cells. We have added this information to the manuscript. Consistent with our agnostic approach (see point 1), we have chosen not to include specific splice variant information, as we cannot guarantee their detectability under the conditions used. 5. Lot numbers for antibodies: Thank you for pointing this out. The lot numbers were already included in Table 1 (third column). 6. Replicates and reproducibility: We have added a new “Limitations” section preceding the Methods to address this point more explicitly. In brief, replicates are addressed indirectly through use of cross-licensed antibodies from different manufacturers. Experiments are standardized using master mixes and consistent protocols. Where signal is absent, repeat experiments are conducted. For IF, dual concentrations are tested to assess performance. 7–9. Choice of antibody for WB of IPs and secondary antibodies used: We have revised the manuscript to clarify that MA5-27542 (a mouse monoclonal) was selected for immunoblotting of immunoprecipitates to avoid species overlap, as most tested IP antibodies are rabbit-derived. Using the same antibody across all IP samples facilitates direct performance comparison. The correct secondary antibodies were used in all applications, and their identities have been specified in the revised manuscript. 10. IF image display and labeling: Thank you for noting this. The legend now correctly indicates that separate (not merged) grayscale images are shown. We have retained grayscale display, consistent with microscopy best practices for visual clarity. 11. Use of a single cell line: We agree that this is a limitation. SCD1 is broadly expressed and considered an essential gene. HeLa cells express SCD1 at a level slightly below the median across cancer cell lines analyzed by DepMap (6.7 vs. 7.0 TPM). We now highlight this limitation and rationale in the revised manuscript. 12. Clarification of high-quality antibodies and application-specific summary: While we avoid drawing formal conclusions on antibody performance, we do indicate those used in key figures (e.g., Figures 2 and 3). As noted, interpretation is left to the end user, with guidance provided in the editorial accompanying our gateway (DOI: 10.12688/f1000research.141719.1). We also added Table 3 that guides researchers interpret validation data. Minor comments: Terminology (IF vs ICC): The manuscript now includes both terms for clarity. UniProt ID in title: We retained the UniProt ID in the title due to the high variability in protein names, which helps ensure accurate identification. Clarification of endogenous expression: The relevant section has been revised to clarify that all expression pertains to endogenous protein levels. Mycoplasma testing and cell line authentication: A statement regarding mycoplasma testing and cell line authentication has been added to the Methods section. Epitope information: As a public initiative, our focus is to provide KO-based, standardized antibody validation across key applications. While we value reviewer and community suggestions, incorporating additional information would introduce burdens that could limit the scalability and efficiency of the initiative. Exposure times for WB: We have chosen not to include exposure times, as they vary with protein loading, detection systems, and antibody dilutions, which would limit reproducibility across labs. Antibody label in IP figure: We have decided to keep the labelling as it is. Cell quantification in IF: We now specify that at least 215 wild-type and knockout cells were quantified, addressing the reviewer’s concern. Magnification in IF figures: The magnification is specified in the figure legend (scale bar = 10 µm). Related antibodies (clones): We now explicitly state that ab19862 and MA5-27542 originate from the same clone. Clarification of renewable antibodies: We agree and have revised the statement in question. Since only two unique renewable antibodies were tested, we removed the word “several.” Grammar and spelling corrections: All grammar, spelling, and punctuation issues flagged have been addressed in the revised manuscript. Citation of protocol paper: Ayoubi et al., Nature Protocols (2024) has been properly cited. We are grateful for your insightful comments, which have helped us improve the clarity and rigor of this antibody characterization guide. Sincerely, Carl Laflamme (on behalf of all co-authors) Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 11 Sep 2025 Carl Laflamme , Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, Montreal, Canada 11 Sep 2025 Author Response Dear Dr. Williams and Dr. Holm, Thank you for your thoughtful review and constructive comments on our manuscript. We appreciate your recognition of the value and reproducibility aims of ... Continue reading Dear Dr. Williams and Dr. Holm, Thank you for your thoughtful review and constructive comments on our manuscript. We appreciate your recognition of the value and reproducibility aims of the YCharOS initiative, and we have revised the manuscript accordingly. Please find below our responses to your major and minor comments: Major comments: 1. Inclusion of basic protein information (subcellular localization, molecular weight, splice variants): We appreciate the suggestion. However, we deliberately avoid including expected localization or molecular weight data to maintain an agnostic and unbiased evaluation of antibody performance. Including such information can lead to confirmation bias, particularly when unreliable antibodies have historically led to incorrect assumptions about localization or molecular weight. While the theoretical molecular weight of SCD1 is provided in the Figure 1 legend, we acknowledge that SDS-PAGE migration can deviate due to post-translational modifications or proteolytic processing. 2. Rationale for selecting SCD1 as a target: The study was funded by the Simons Foundation Autism Research Initiative (SFARI), which surveyed its community to identify autism-relevant proteins lacking high-quality research tools. SCD1 was among seven prioritized targets identified for systematic antibody validation. This supports the relevance and urgency of the work. 3. Use of the term “most commercially available antibodies”: We agree with the reviewer. The sentence has been revised to refer specifically to “renewable antibodies,” which aligns with the language used in our previous publications. We also note that polyclonal antibodies are often cross-licensed under different catalog numbers, complicating estimates of the actual number of unique reagents. 4. Confirmation of HeLa WT status and splice variants: According to DepMap.org, the SCD1 gene is not mutated in HeLa cells. We have added this information to the manuscript. Consistent with our agnostic approach (see point 1), we have chosen not to include specific splice variant information, as we cannot guarantee their detectability under the conditions used. 5. Lot numbers for antibodies: Thank you for pointing this out. The lot numbers were already included in Table 1 (third column). 6. Replicates and reproducibility: We have added a new “Limitations” section preceding the Methods to address this point more explicitly. In brief, replicates are addressed indirectly through use of cross-licensed antibodies from different manufacturers. Experiments are standardized using master mixes and consistent protocols. Where signal is absent, repeat experiments are conducted. For IF, dual concentrations are tested to assess performance. 7–9. Choice of antibody for WB of IPs and secondary antibodies used: We have revised the manuscript to clarify that MA5-27542 (a mouse monoclonal) was selected for immunoblotting of immunoprecipitates to avoid species overlap, as most tested IP antibodies are rabbit-derived. Using the same antibody across all IP samples facilitates direct performance comparison. The correct secondary antibodies were used in all applications, and their identities have been specified in the revised manuscript. 10. IF image display and labeling: Thank you for noting this. The legend now correctly indicates that separate (not merged) grayscale images are shown. We have retained grayscale display, consistent with microscopy best practices for visual clarity. 11. Use of a single cell line: We agree that this is a limitation. SCD1 is broadly expressed and considered an essential gene. HeLa cells express SCD1 at a level slightly below the median across cancer cell lines analyzed by DepMap (6.7 vs. 7.0 TPM). We now highlight this limitation and rationale in the revised manuscript. 12. Clarification of high-quality antibodies and application-specific summary: While we avoid drawing formal conclusions on antibody performance, we do indicate those used in key figures (e.g., Figures 2 and 3). As noted, interpretation is left to the end user, with guidance provided in the editorial accompanying our gateway (DOI: 10.12688/f1000research.141719.1). We also added Table 3 that guides researchers interpret validation data. Minor comments: Terminology (IF vs ICC): The manuscript now includes both terms for clarity. UniProt ID in title: We retained the UniProt ID in the title due to the high variability in protein names, which helps ensure accurate identification. Clarification of endogenous expression: The relevant section has been revised to clarify that all expression pertains to endogenous protein levels. Mycoplasma testing and cell line authentication: A statement regarding mycoplasma testing and cell line authentication has been added to the Methods section. Epitope information: As a public initiative, our focus is to provide KO-based, standardized antibody validation across key applications. While we value reviewer and community suggestions, incorporating additional information would introduce burdens that could limit the scalability and efficiency of the initiative. Exposure times for WB: We have chosen not to include exposure times, as they vary with protein loading, detection systems, and antibody dilutions, which would limit reproducibility across labs. Antibody label in IP figure: We have decided to keep the labelling as it is. Cell quantification in IF: We now specify that at least 215 wild-type and knockout cells were quantified, addressing the reviewer’s concern. Magnification in IF figures: The magnification is specified in the figure legend (scale bar = 10 µm). Related antibodies (clones): We now explicitly state that ab19862 and MA5-27542 originate from the same clone. Clarification of renewable antibodies: We agree and have revised the statement in question. Since only two unique renewable antibodies were tested, we removed the word “several.” Grammar and spelling corrections: All grammar, spelling, and punctuation issues flagged have been addressed in the revised manuscript. Citation of protocol paper: Ayoubi et al., Nature Protocols (2024) has been properly cited. We are grateful for your insightful comments, which have helped us improve the clarity and rigor of this antibody characterization guide. Sincerely, Carl Laflamme (on behalf of all co-authors) Dear Dr. Williams and Dr. Holm, Thank you for your thoughtful review and constructive comments on our manuscript. We appreciate your recognition of the value and reproducibility aims of the YCharOS initiative, and we have revised the manuscript accordingly. Please find below our responses to your major and minor comments: Major comments: 1. Inclusion of basic protein information (subcellular localization, molecular weight, splice variants): We appreciate the suggestion. However, we deliberately avoid including expected localization or molecular weight data to maintain an agnostic and unbiased evaluation of antibody performance. Including such information can lead to confirmation bias, particularly when unreliable antibodies have historically led to incorrect assumptions about localization or molecular weight. While the theoretical molecular weight of SCD1 is provided in the Figure 1 legend, we acknowledge that SDS-PAGE migration can deviate due to post-translational modifications or proteolytic processing. 2. Rationale for selecting SCD1 as a target: The study was funded by the Simons Foundation Autism Research Initiative (SFARI), which surveyed its community to identify autism-relevant proteins lacking high-quality research tools. SCD1 was among seven prioritized targets identified for systematic antibody validation. This supports the relevance and urgency of the work. 3. Use of the term “most commercially available antibodies”: We agree with the reviewer. The sentence has been revised to refer specifically to “renewable antibodies,” which aligns with the language used in our previous publications. We also note that polyclonal antibodies are often cross-licensed under different catalog numbers, complicating estimates of the actual number of unique reagents. 4. Confirmation of HeLa WT status and splice variants: According to DepMap.org, the SCD1 gene is not mutated in HeLa cells. We have added this information to the manuscript. Consistent with our agnostic approach (see point 1), we have chosen not to include specific splice variant information, as we cannot guarantee their detectability under the conditions used. 5. Lot numbers for antibodies: Thank you for pointing this out. The lot numbers were already included in Table 1 (third column). 6. Replicates and reproducibility: We have added a new “Limitations” section preceding the Methods to address this point more explicitly. In brief, replicates are addressed indirectly through use of cross-licensed antibodies from different manufacturers. Experiments are standardized using master mixes and consistent protocols. Where signal is absent, repeat experiments are conducted. For IF, dual concentrations are tested to assess performance. 7–9. Choice of antibody for WB of IPs and secondary antibodies used: We have revised the manuscript to clarify that MA5-27542 (a mouse monoclonal) was selected for immunoblotting of immunoprecipitates to avoid species overlap, as most tested IP antibodies are rabbit-derived. Using the same antibody across all IP samples facilitates direct performance comparison. The correct secondary antibodies were used in all applications, and their identities have been specified in the revised manuscript. 10. IF image display and labeling: Thank you for noting this. The legend now correctly indicates that separate (not merged) grayscale images are shown. We have retained grayscale display, consistent with microscopy best practices for visual clarity. 11. Use of a single cell line: We agree that this is a limitation. SCD1 is broadly expressed and considered an essential gene. HeLa cells express SCD1 at a level slightly below the median across cancer cell lines analyzed by DepMap (6.7 vs. 7.0 TPM). We now highlight this limitation and rationale in the revised manuscript. 12. Clarification of high-quality antibodies and application-specific summary: While we avoid drawing formal conclusions on antibody performance, we do indicate those used in key figures (e.g., Figures 2 and 3). As noted, interpretation is left to the end user, with guidance provided in the editorial accompanying our gateway (DOI: 10.12688/f1000research.141719.1). We also added Table 3 that guides researchers interpret validation data. Minor comments: Terminology (IF vs ICC): The manuscript now includes both terms for clarity. UniProt ID in title: We retained the UniProt ID in the title due to the high variability in protein names, which helps ensure accurate identification. Clarification of endogenous expression: The relevant section has been revised to clarify that all expression pertains to endogenous protein levels. Mycoplasma testing and cell line authentication: A statement regarding mycoplasma testing and cell line authentication has been added to the Methods section. Epitope information: As a public initiative, our focus is to provide KO-based, standardized antibody validation across key applications. While we value reviewer and community suggestions, incorporating additional information would introduce burdens that could limit the scalability and efficiency of the initiative. Exposure times for WB: We have chosen not to include exposure times, as they vary with protein loading, detection systems, and antibody dilutions, which would limit reproducibility across labs. Antibody label in IP figure: We have decided to keep the labelling as it is. Cell quantification in IF: We now specify that at least 215 wild-type and knockout cells were quantified, addressing the reviewer’s concern. Magnification in IF figures: The magnification is specified in the figure legend (scale bar = 10 µm). Related antibodies (clones): We now explicitly state that ab19862 and MA5-27542 originate from the same clone. Clarification of renewable antibodies: We agree and have revised the statement in question. Since only two unique renewable antibodies were tested, we removed the word “several.” Grammar and spelling corrections: All grammar, spelling, and punctuation issues flagged have been addressed in the revised manuscript. Citation of protocol paper: Ayoubi et al., Nature Protocols (2024) has been properly cited. We are grateful for your insightful comments, which have helped us improve the clarity and rigor of this antibody characterization guide. Sincerely, Carl Laflamme (on behalf of all co-authors) Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 02 Jan 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 3 Version 2 (revision) 13 Jun 25 read Version 1 02 Jan 25 read read Cecilia Williams , KTH Royal Institute of Technology,, Stockholm, Sweden Matilda Holm , KTH Royal Institute of Technology (Ringgold ID: 7655), Stockholm, Sweden Michael L. Garelja , University of Otago, Dunedin, New Zealand Deborah Moshinsky , Institute for Protein Innovation, Boston, USA Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 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. 10 Jul 2025 | for Version 2 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 9 commercially available antibodies to SCD1 (stearoyl-CoA desaturase) for activity in Western Blot, Immunoprecipitation (IP) and immunofluorescence (IF). They first found a cell line expressing the protein and generated a knockout version. Then they utilized standardized, openly available protocols for Western, IP and IF. The results were clear and the methodology easy to follow. Table 3 is useful to assist in the interpretation of the data. However, for the Western Blot examples in Table 3, the situation where multiple bands including the target are present in the WT and multiple bands but lacking the target is present in the KO is labeled as a 'successful antibody.' I recommend that this be clarified, as one could argue that an antibody that detects it's target along with other proteins is non-specific and therefore not a 'successful' antibody. Perhaps successful but non-selective or another qualifying term would be more suitable. Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Antibody 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 Stearoyl-CoA desaturase (SCD1) (UniProt ID: O00767) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :10 ( https://doi.org/10.5256/f1000research.183557.r392564) 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/14-10/v2#referee-response-392564 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Garelja M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 05 Feb 2025 | for Version 1 Michael L. Garelja , University of Otago, Dunedin, New Zealand 0 Views copyright © 2025 Garelja M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) 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 This manuscript, aiming to investigate the utility of a range of SCD1 antibodies is extremely important, and I applaud the initiative. Identification of antibodies that can correctly detect their target is of incredible importance to the entire field and highlighting that not all antibodies work as advertised is critical to ensuring that researchers can perform robust science. Points that need be addressed for this work to be indexed. Are there RRID’s associated with these antibodies? This applies for both the primary antibodies, and the secondary antibodies. Secondary antibodies also require lot numbers. As antibody concentrations can change between batches, it would be more informative to list the concentrations used in the experiments rather than the dilutions. A summary table to show how the different antibodies work under different experimental techniques would be extremely useful. I understand that the resource cannot make recommendations, but having a summary of how each antibody would help others interpret results. Are there any known splice variants of this target? There is a consistent band of approximately 25 kDa, that also seems to be lower when knocked out. Any insight as to why the detected band differed from the estimated molecular weight would also be useful. Has this particular ladder been compared to other ladders to ensure that it runs as expected under these experimental conditions? Further information for discussion. The researchers have used a cell-line which highly expresses the target. Is it possible to work with cells that have lower expression levels? If not, these needs to be addressed as a limitation. Testing the panel of “useful” antibodies against more cell lines with variable SCD1 expression to show a limit of detection would be of high utility. Listing the epitopes that the antibodies were raised against would be of high value. One of the pillars of antibody validation is to use two antibodies that have been raised against different epitopes, so having that information readily available would help researchers in their mission to perform good science. The paper should acknowledge that there may be methodological optimizations that could improve antibody function. Figure 3 – is it possible to show to green and red stained cells in a separate panel? The outlines are useful, but showing that the cells have similar morphologies (or not!) would be interesting in the context of SCD1 biology. Why were these antibodies chosen, as opposed to the wider range of antibodies available? This is not a criticism, as the work is valuable regardless, but some information as to how these antibodies were selected would be interesting. 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 Competing Interests No competing interests were disclosed. Reviewer Expertise CGRP, migraine, pharmacology 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 (1) Author Response 10 Sep 2025 Carl Laflamme, Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, Montreal, Canada Dear Dr. Garelja, Thank you for your encouraging words and thoughtful suggestions. We greatly appreciate your support of the YCharOS initiative and your detailed feedback, which helped us further strengthen the manuscript. Please find our responses to your comments below: 1. RRIDs for antibodies: We have carefully assigned RRIDs to all primary antibodies. In this report, polyclonal secondary antibodies were used. However, we are transitioning toward the exclusive use of recombinant secondary antibodies, and RRIDs will be included for those reagents in future studies. 2. Reporting concentrations vs. dilutions: We understand the rationale for suggesting antibody concentrations. However, to maintain consistency with manufacturer guidelines and accommodate proprietary formulations (where concentrations are not disclosed), we report antibody usage as dilutions, as recommended by most vendors. 3. Summary of antibody performance: We have added a new Table (Table 3) to provide a schematic summary of antibody performance across applications. While we do not rank antibodies, this overview will help users interpret the data more efficiently. 4. Unexpected SDS-PAGE migration / lower molecular weight band: As this is an agnostic antibody validation effort, we refrain from interpreting specific bands. Nonetheless, we agree this is a valuable point for discussion. Proteins may migrate differently than expected on SDS-PAGE due to post-translational modifications, unusual amino acid composition, protein folding, or incomplete denaturation, which can affect SDS binding and alter migration behavior. The consistent ~25 kDa band and its reduction in the knockout sample may represent a splice variant, degradation product, or non-specific interaction. We have included this discussion in the revised manuscript. Minor Comments 1. Use of a high-expressing cell line: We agree this is a limitation. SCD1 is broadly expressed and considered an essential gene. HeLa cells express SCD1 slightly below the median level across cancer cell lines (6.7 vs. 7.0 TPM; DepMap data). We now highlight this rationale and limitation explicitly in the revised manuscript. 2. Epitope: As a public initiative, our focus is to provide KO-based, standardized antibody validation across key applications. While we value reviewer and community suggestions, incorporating additional information would introduce burdens that could limit the scalability and efficiency of the initiative. 3. Methodological optimization: We have acknowledged in the revised text that further optimization of protocols may improve antibody performance and encourage researchers to adapt conditions to their specific needs. 4. Raw data accessibility: All raw data files are openly available via Zenodo, as described in the Data Availability section of the manuscript. 5. Antibody selection criteria: The antibodies included in this study were provided free of charge by participating manufacturers. Selection was based on availability, vendor prioritization, and a preference for renewable reagents where possible. We have clarified this in the revised manuscript. Thank you for your comments, which have helped us improve the clarity and rigor of this antibody characterization guide. Sincerely, Carl Laflamme (on behalf of all co-authors) View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Garelja ML. Peer Review Report For: A guide to selecting high-performing antibodies for Stearoyl-CoA desaturase (SCD1) (UniProt ID: O00767) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :10 ( https://doi.org/10.5256/f1000research.176074.r358436) 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/14-10/v1#referee-response-358436 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Williams C et al. 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. 17 Jan 2025 | for Version 1 Cecilia Williams , KTH Royal Institute of Technology,, Stockholm, Sweden Matilda Holm , Protein Science, KTH Royal Institute of Technology (Ringgold ID: 7655), Stockholm, Stockholm County, Sweden 0 Views copyright © 2025 Williams C et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved 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 The data note by Ruiz Moleon et al. provides a high-quality antibody validation guide for the protein SCD1. The guide is part of the YCharOS initiative that uses isogenic knockout cell lines and multiple antibody-dependent assays to investigate antibody specificity and selectivity in a structured manner. It is an important effort that can improve the reproducibility of biomedical research, and this guide is highly useful for researchers focusing on SCD1. The authors show that 3-4 out of 9 antibodies do not work in most or any of the assays, whereas the rest are more or less well-performing, using the cells and conditions applied in the protocol. This is critical information that will be of high value to the research community. For enhanced usability, clarification, and stringency of the guide and to facilitate the interpretation and reproducibility, I have the below recommendations. Major comments The introduction would benefit from including basic information regarding the protein, as this would facilitate the interpretation of the data. Such as the known or predicted subcellular localization of SCD1, its calculated molecular weight, and if any splice variants are known. The rationale, as stated (p.3, 2 nd paragraph: “ Mechanistic studies would be facilitated with the availability of high-quality SCD1 antibodies ”) should be better supported. Is there a documented lack of high-quality SCD1 antibodies? Or, is this an assumption based on the general problem with the reproducibility of antibody-based research? The sentence “ using most commercially available antibodies against the corresponding protein” (p. 3, 3 rd paragraph) is likely not correct. Perhaps the authors intended to state “ renewable antibodies” as the initiative states in other publications. In this guide, specifically, the authors include 9 commercial (renewable and non-renewable) SCD1 antibodies, but there are as many as 274 antibodies from 32 providers directed towards human SCD1 listed in Antibodypedia. A statement of whether the SCD1 gene in the cells (HeLa WT) has indeed been confirmed to be wild type (i.e., not mutated) should be added. It would also be useful to include which (if any) splice variant transcripts are expressed in these cells. Table 1: The lot (or batch) numbers for the antibodies must be listed. This is essential information and is especially important in an antibody validation guide. It should be stated whether replicate experiments were performed. It is unclear how the antibody used for the western blot of the IPs was chosen (p. 3, 2 nd last paragraph: “ a specific antibody was chosen ”). Were both specificity and selectivity considered? Were the species the antibodies were generated in used as criteria (to avoid detection of heavy and light chains in the next step)? Or was the selection random out of the sufficiently specific antibodies? If so, how many and which antibodies were deemed to be sufficiently specific? Fig 2: Considering that antibodies from different species appear specific in WB, it seems like a missed opportunity to not take species into account to generate clearer western blot data of the IPs (i.e., avoiding additional bands of heavy and light antibody chains, such as when using mouse-generated antibodies in both IP and WB and detecting them with an anti-mouse secondary antibody). Fig. 1 and 2: It should be specified which secondary antibody was used in the western blots. Fig. 3: The legend statement that “ Representative images of the merged blue and red (grayscale) channels are shown. ” appears incorrect. The blue (DAPI) is shown separately from the red (antibody staining); they are not merged in the figure. It would also be clearer if the images showed the actual colors (blue for DAPI and red for SCD1 antibody) instead of grey for both. Only one cell line is used, which is a limitation of the initiative. Considering that HeLa cells express unusually high SCD1 levels (as stated in the text), the antibodies may work less well at lower SCD1 levels. They could also bind to other proteins expressed in other cells but not HeLa. It would be useful if the antibodies (the ones deemed to be of high quality) were additionally analyzed by western blot on a panel of cell lines with varying mRNA expression of SCD1 . This would indicate their performance at different levels of the target protein and in more than one cell line. Although the authors state that they do not engage in result analysis, they do (to select a specific antibody for WB of the IP, and they conclude that several antibodies successfully detect SCD1), but they refrain from clearly stating which these antibodies are. A table, or a statement, summarizing which antibodies bind intended and unintended targets in the different assays (under the specific conditions used) would allow greater usability, including for researchers starting their work (such as postdocs and PhD students). This would also facilitate an overview of the application-dependent performance (again, under the specific conditions used). Minor comments: The assay immunofluorescence (IF) is typically used for tissue analysis. For analysis of cells and cell lines, as is the case here, the term immunocytochemistry is better suited. UniProt ID may not need to be stated in the title (could be moved to abstract or introduction) p.3, 3 rd paragraph: It should be clarified if the target protein expression relates specifically to endogenous expression or whether cell lines with recombinant expression were included. A statement about mycoplasma testing and cell line authentication should be included. It would be beneficial to include which epitope each antibody is directed toward (or raised against). Fig. 1 and 2: The exposure time(s) applied for the western blots could be included. Fig 2: Although the figure legend clearly states which antibody was used for WB of all IPs, the figure itself gives the impression that it may have been used only for the two indicated IPs. It might be better to remove this from the image or place it differently. p. 5, first paragraph: “ Quantification of ... in hundreds of WT and KO cells ” is vague. The number (or range) of actually quantified cells should be stated. Fig. 3: The figure legend should specify which magnification was used for the IF images. It would be beneficial to highlight that the antibodies ab19862 and MA-27542 are from the same clone and should be the same. It would be pedagogical to place them side by side. Page 5, final paragraph: “ Several high-quality and renewable antibodies that successfully detect SCD1 were identified in all applications”. As only 3 of the tested antibodies were renewable (monoclonal or recombinant), and two of them were from the same clone (= same antibody), the word “several” seems a stretch. The word “renewable” should probably be removed. Several spelling/grammar errors (p.3 a “to” too much p.3, p. 3 “were run “ instead of “were ran”, wrongly placed commas, etc.) The published method protocol Ayoubi et al., Nature Protocol (2024) should likely be referenced. Author roles: It is not stated who drafted the manuscript, nor if all authors commented on it. 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? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise nuclear receptors, transcriptomes, antibody validation, cancer We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 11 Sep 2025 Carl Laflamme, Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, Montreal, Canada Dear Dr. Williams and Dr. Holm, Thank you for your thoughtful review and constructive comments on our manuscript. We appreciate your recognition of the value and reproducibility aims of the YCharOS initiative, and we have revised the manuscript accordingly. Please find below our responses to your major and minor comments: Major comments: 1. Inclusion of basic protein information (subcellular localization, molecular weight, splice variants): We appreciate the suggestion. However, we deliberately avoid including expected localization or molecular weight data to maintain an agnostic and unbiased evaluation of antibody performance. Including such information can lead to confirmation bias, particularly when unreliable antibodies have historically led to incorrect assumptions about localization or molecular weight. While the theoretical molecular weight of SCD1 is provided in the Figure 1 legend, we acknowledge that SDS-PAGE migration can deviate due to post-translational modifications or proteolytic processing. 2. Rationale for selecting SCD1 as a target: The study was funded by the Simons Foundation Autism Research Initiative (SFARI), which surveyed its community to identify autism-relevant proteins lacking high-quality research tools. SCD1 was among seven prioritized targets identified for systematic antibody validation. This supports the relevance and urgency of the work. 3. Use of the term “most commercially available antibodies”: We agree with the reviewer. The sentence has been revised to refer specifically to “renewable antibodies,” which aligns with the language used in our previous publications. We also note that polyclonal antibodies are often cross-licensed under different catalog numbers, complicating estimates of the actual number of unique reagents. 4. Confirmation of HeLa WT status and splice variants: According to DepMap.org, the SCD1 gene is not mutated in HeLa cells. We have added this information to the manuscript. Consistent with our agnostic approach (see point 1), we have chosen not to include specific splice variant information, as we cannot guarantee their detectability under the conditions used. 5. Lot numbers for antibodies: Thank you for pointing this out. The lot numbers were already included in Table 1 (third column). 6. Replicates and reproducibility: We have added a new “Limitations” section preceding the Methods to address this point more explicitly. In brief, replicates are addressed indirectly through use of cross-licensed antibodies from different manufacturers. Experiments are standardized using master mixes and consistent protocols. Where signal is absent, repeat experiments are conducted. For IF, dual concentrations are tested to assess performance. 7–9. Choice of antibody for WB of IPs and secondary antibodies used: We have revised the manuscript to clarify that MA5-27542 (a mouse monoclonal) was selected for immunoblotting of immunoprecipitates to avoid species overlap, as most tested IP antibodies are rabbit-derived. Using the same antibody across all IP samples facilitates direct performance comparison. The correct secondary antibodies were used in all applications, and their identities have been specified in the revised manuscript. 10. IF image display and labeling: Thank you for noting this. The legend now correctly indicates that separate (not merged) grayscale images are shown. We have retained grayscale display, consistent with microscopy best practices for visual clarity. 11. Use of a single cell line: We agree that this is a limitation. SCD1 is broadly expressed and considered an essential gene. HeLa cells express SCD1 at a level slightly below the median across cancer cell lines analyzed by DepMap (6.7 vs. 7.0 TPM). We now highlight this limitation and rationale in the revised manuscript. 12. Clarification of high-quality antibodies and application-specific summary: While we avoid drawing formal conclusions on antibody performance, we do indicate those used in key figures (e.g., Figures 2 and 3). As noted, interpretation is left to the end user, with guidance provided in the editorial accompanying our gateway (DOI: 10.12688/f1000research.141719.1). We also added Table 3 that guides researchers interpret validation data. Minor comments: Terminology (IF vs ICC): The manuscript now includes both terms for clarity. UniProt ID in title: We retained the UniProt ID in the title due to the high variability in protein names, which helps ensure accurate identification. Clarification of endogenous expression: The relevant section has been revised to clarify that all expression pertains to endogenous protein levels. Mycoplasma testing and cell line authentication: A statement regarding mycoplasma testing and cell line authentication has been added to the Methods section. Epitope information: As a public initiative, our focus is to provide KO-based, standardized antibody validation across key applications. While we value reviewer and community suggestions, incorporating additional information would introduce burdens that could limit the scalability and efficiency of the initiative. Exposure times for WB: We have chosen not to include exposure times, as they vary with protein loading, detection systems, and antibody dilutions, which would limit reproducibility across labs. Antibody label in IP figure: We have decided to keep the labelling as it is. Cell quantification in IF: We now specify that at least 215 wild-type and knockout cells were quantified, addressing the reviewer’s concern. Magnification in IF figures: The magnification is specified in the figure legend (scale bar = 10 µm). Related antibodies (clones): We now explicitly state that ab19862 and MA5-27542 originate from the same clone. Clarification of renewable antibodies: We agree and have revised the statement in question. Since only two unique renewable antibodies were tested, we removed the word “several.” Grammar and spelling corrections: All grammar, spelling, and punctuation issues flagged have been addressed in the revised manuscript. Citation of protocol paper: Ayoubi et al., Nature Protocols (2024) has been properly cited. We are grateful for your insightful comments, which have helped us improve the clarity and rigor of this antibody characterization guide. Sincerely, Carl Laflamme (on behalf of all co-authors) View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Williams C and Holm M. Peer Review Report For: A guide to selecting high-performing antibodies for Stearoyl-CoA desaturase (SCD1) (UniProt ID: O00767) for use in western blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :10 ( https://doi.org/10.5256/f1000research.176074.r355980) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. 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