A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence

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TGM2 is involved in various diseases, either in a protective or contributory manner, making it a crucial protein to study and determine its therapeutic potential. Identifying high-performing TGM2 antibodies would facilitate these investigations. Here we have characterized seventeen TGM2 commercial antibodies for western blot and sixteen for immunoprecipitation, and immunofluorescence. The implemented standardized experimental protocol is based on comparing read-outs in knockout cell lines against their isogenic parental controls. This study is part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs." } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/13-481/v2", "name": "A guide to selecting high-performing antibodies for Protein-glutamine..." } } ] } Home Browse A guide to selecting high-performing antibodies for Protein-glutamine... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Ayoubi R, Fotouhi M, Alende C et al. A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved, 1 approved with reservations] . F1000Research 2024, 13 :481 ( https://doi.org/10.12688/f1000research.150684.2 ) 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 Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved, 1 approved with reservations] Riham Ayoubi 1 , Maryam Fotouhi 1 , Charles Alende https://orcid.org/0009-0005-4611-6134 1 , [...] Sara González Bolívar https://orcid.org/0000-0002-4299-8281 1 , Kathleen Southern https://orcid.org/0000-0002-4125-3608 1 , Carl Laflamme https://orcid.org/0000-0002-4125-3608 1 , Neuro/SGC/EDDU collaborative group , ABIF consortium Riham Ayoubi 1 , Maryam Fotouhi 1 , [...] Charles Alende https://orcid.org/0009-0005-4611-6134 1 , Sara González Bolívar https://orcid.org/0000-0002-4299-8281 1 , Kathleen Southern https://orcid.org/0000-0002-4125-3608 1 , Carl Laflamme https://orcid.org/0000-0002-4125-3608 1 , Neuro/SGC/EDDU collaborative group , ABIF consortium PUBLISHED 30 Jul 2024 Author details Author details 1 Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Québec, H3A 2B4, Canada Riham Ayoubi Roles: Formal Analysis, Investigation, Methodology, Validation, Visualization Maryam Fotouhi Roles: Investigation, Validation, Visualization Charles Alende Roles: Investigation, Validation, Visualization Sara González Bolívar Roles: Investigation, Validation, Visualization Kathleen Southern Roles: Writing – Original Draft Preparation, Writing – Review & Editing Carl Laflamme Roles: Conceptualization, Formal Analysis, Funding Acquisition, Methodology, Resources, Validation, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the YCharOS (Antibody Characterization through Open Science) gateway. Abstract Protein-glutamine gamma-glutamyltransferase 2 (TGM2) is a Ca 2+ dependent enzyme that catalyzes transglutaminase cross-linking modifications. TGM2 is involved in various diseases, either in a protective or contributory manner, making it a crucial protein to study and determine its therapeutic potential. Identifying high-performing TGM2 antibodies would facilitate these investigations. Here we have characterized seventeen TGM2 commercial antibodies for western blot and sixteen for immunoprecipitation, and immunofluorescence. The implemented standardized experimental protocol is based on comparing read-outs in knockout cell lines against their isogenic parental controls. This study is part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs. READ ALL READ LESS Keywords Uniprot ID P21980, TGM2, , TGM, Protein-glutamine gamma-glutamyltransferase 2, antibody characterization, antibody validation, western blot, immunoprecipitation, immunofluorescence Corresponding Author(s) Carl Laflamme ( [email protected] ) Close Corresponding author: Carl Laflamme Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - ABCD antibodies - Abcam - Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific. Grant information: This work was supported by the Michael J. Fox Foundation for Parkinson’s Research (MJFF) (grant no. 18331). This work was also supported by a grant from the Canadian Institutes of Health Research Foundation (grant no. FDN154305), the Government of Canada through Genome Canada, Genome Quebec and Ontario Genomics (grant no. OGI-210). RA is supported by Mitacs fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2024 Ayoubi R et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Ayoubi R, Fotouhi M, Alende C et al. A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved, 1 approved with reservations] . F1000Research 2024, 13 :481 ( https://doi.org/10.12688/f1000research.150684.2 ) First published: 17 May 2024, 13 :481 ( https://doi.org/10.12688/f1000research.150684.1 ) Latest published: 30 Jul 2024, 13 :481 ( https://doi.org/10.12688/f1000research.150684.2 ) Revised Amendments from Version 1 In version 2 of this article, we have clearly defined how the A549 WT and TGM2 KO cells were prepared and collected as lysates (medium-free) or on culture medium prior to western blot evaluation. Additionally, two columns have been added to Table 2 to indicate the immunogenic region of the antibodies as well as the number of citations for each antibody, as listed on the manufacturer catalogs and CiteAb.com, respectively. In version 2 of this article, we have clearly defined how the A549 WT and TGM2 KO cells were prepared and collected as lysates (medium-free) or on culture medium prior to western blot evaluation. Additionally, two columns have been added to Table 2 to indicate the immunogenic region of the antibodies as well as the number of citations for each antibody, as listed on the manufacturer catalogs and CiteAb.com, respectively. See the authors' detailed response to the review by Michael A Rieger See the authors' detailed response to the review by Nicoletta Bianchi READ REVIEWER RESPONSES Introduction Protein-glutamine gamma glutamyltransferase 2 (TGM2) belongs to the transglutaminase family of Ca 2+ dependent enzymes, regulating various cellular processes, including cell differentiation, growth and apoptosis. 1 – 3 Encoded by TGM2 gene, TGM2 protein exhibits its function through Ca 2+ -dependent cross-linking of substrates, thereby modulating their activity. 1 TGM2’s catalytic activity is dependent on guanine nucleotides and Ca 2+ binding. 4 Both GTP and/or GDP act as negative regulators of TGM2, inducing a conformational change upon binding, inhibiting its cross-linking activity (closed form). Conversely, Ca 2+ binding prompts a conformational change to induce TGM2 activity (open form). 5 – 9 Alteration and regulation of TGM2’s activity is associated with the pathogenesis of various diseases including cancer, neurodegeneration, fibrosis, inflammatory, autoimmune disorders and liver diseases. 10 – 13 Increased TGM2 mRNA transcripts, resulting in elevated transaminase enzymatic activity, have been associated with neurodegenerative mechanism observed in Parkinson’s disease, Alzheimer’s disease and Huntington’s disease. 14 , 15 Given that α-synuclein serves as a common substrate for TGM2, elevated activity of TGM2 results in the formation of soluble aggregates as well as insoluble inclusions; distinctive features of Parkinson’s disease pathogenesis. 15 – 18 Regulating TGM2 activity using inhibitors could positively affect the human diseases in which TGM2 is implicated. 19 , 20 Identifying high-quality antibodies would accelerate TGM2 research and its potential as a pharmacological target. This research is part of a broader collaborative initiative in which academics, funders and commercial antibody manufacturers are working together to address antibody reproducibility issues by characterizing commercial antibodies for human proteins using standardized protocols, and openly sharing the data. 21 – 23 Here, we evaluated the performance of seventeen commercially-available antibodies for TGM2 for use in western blot, and sixteen for immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of TGM2 properties and function. The platform for antibody characterization used to carry out this study was approved by a committee of industry and academic researchers, whose members are mentioned in the competing interest section. It consists of first identifying appropriate human cell lines, development/contribution of equivalent knockout cell lines and finally following antibody characterization procedures on commonly used commercial antibodies. The standardized consensus antibody characterization protocols are openly available on Protocol Exchange, DOI: 10.21203/rs.3.pex-2607/v1 . 24 Results and discussion Our standard protocol involves comparing readouts from wild-type (WT) and knockout (KO) cells. 25 , 26 The first step is to identify a cell line(s) that expresses sufficient levels of a given protein to generate a measurable signal. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log 2 (transcripts per million “TPM” + 1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). Commercially available A549 cells expressed the TGM2 transcript at RNA levels above the average range of cancer cells analyzed. Parental and TGM2 KO A549 cells were obtained from Abcam ( Table 1 ). Table 1. Summary of the cell lines used. Institution Catalog number RRID (Cellosaurus) Cell line Species of origin Genotype Abcam ab275463 CVCL_0023 A549 Homo sapiens (Human) WT Abcam ab261876 CVCL_B1I0 A549 Homo sapiens (Human) TGM2 KO For western blot experiments, WT and TGM2 KO cells are collected as lysates or with a serum-free medium, ran on SDS-page, transferred on nitrocellulose membranes, and then probed with all antibodies in parallel ( Figures 1 and 2 ). Figure 1. TGM2 antibody screening by western blot on lysate. Lysates of A549 (WT and TGM2 KO) were prepared and 40 μg of protein were processed for western blot with the indicated TGM2 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Antibody dilution used: ab109121** at 1/1000, ab109200** at 1/10000, ab2386* at 1/500, ab310333** at 1/1000, ab421 at 1/500, ABCD_AI748** at 1/10, A21184** at 1/10000, ARP47471 at 1/500, ARP47472 at 1/500, AF4376 at 1/400, MAB4376* at 1/200, 3557** at 1/500, GTX111702 at 1/500, 15100-1-AP at 1/6000, 68006-1-Ig* at 1/10000, MA5-32819** at 1/500, MA5-12739* at 1/200. Predicted band size: 77 kDa. *Monoclonal antibody, **Recombinant antibody. Figure 2. TGM2 antibody screening by western blot on culture medium. A549 WT and TGM2 KO were cultured in serum free media, and 40 μg of protein from concentrated culture media were processed for western blot with the indicated TGM2 antibodies. The Ponceau stained transfers of each blot are shown. Antibody dilution used: ab109121** at 1/1000, ab109200** at 1/10000, ab2386* at 1/500, ab310333** at 1/1000, ab421 at 1/500, ABCD_AI748** at 1/10, A21184** at 1/10000, ARP47471 at 1/500, ARP47472 at 1/500, AF4376 at 1/400, MAB4376* at 1/200, 3557** at 1/500, GTX111702 at 1/500, 15100-1-AP at 1/6000, 68006-1-Ig* at 1/10000, MA5-32819** at 1/500, MA5-12739* at 1/200. Predicted band size: 77 kDa. *Monoclonal antibody, **Recombinant antibody. As per our standard procedure, we next used the antibodies to immunoprecipitate TGM2 from A549 cell extracts. To evaluate the performance of each antibody, the TGM2 protein was detected in extracts, in each extract unbound to the antibody and corresponding immunoprecipitates (IP) ( Figure 3 ). To detect TGM2, a western blot was performed with an antibody successful under the conditions tested in Figure 1 . Figure 3. TGM2 antibody screening by immunoprecipitation on lysate. A549 lysates were prepared, and immunoprecipitation was performed using 2.0 μg of the indicated TGM2 antibodies pre-coupled to Dynabeads protein A or protein. Samples were washed and processed for western blot with the indicated TGM2 antibody. For western blot, A21184** was used at 1/10000. The Ponceau stained transfers of each blot are shown. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate; n/s=non-specific signal. *Monoclonal antibody, **Recombinant antibody. For immunofluorescence, antibodies were screened using a mosaic strategy, as per our standard procedure. First, A549 WT and TGM2 KO were labelled with different fluorescent dyes in order to distinguish the two cell lines, and TGM2 antibodies were evaluated. Cells were imaged in the same field of view to reduce staining, imaging and image analysis bias ( Figure 4 ). Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested. The images presented in Figure 4 are representative of the results of this analysis. Figure 4. TGM2 antibody screening by immunofluorescence. A549 WT and TGM2 KO cells were labelled with a green or a deep-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio in a 96-well plate with optically clear flat-bottom. Cells were stained with the indicated TGM2 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (identification of WT cells), red (antibody staining) and deep-red (identification of KO cells) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, respectively. When the concentration was not indicated by the supplier, which was the case for all antibodies tested, except ab310333**, ABCD_AI748 and A21184**, we tested antibodies at using the dilutions listed below. At these concentrations, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: ab109121** at 1/1000, ab109200** at 1/300, ab2386* at 1/1000, ab310333** at 1/500, ab421 at 1/1000, ABCD_AI748** at 1/1000, A21184** at 1/1000, ARP47471 at 1/500, ARP47472 at 1/250, AF4376 at 1/100, MAB4376* at 1/100, 3557** at 1/500, GTX111702 at 1/1000, 15100-1-AP at 1/300, 68006-1-Ig* at 1/500, MA5-32819** at 1/1000. Bars = 10 μm. *Monoclonal antibody, **Recombinant antibody. In conclusion, we have screened seventeen TGM2 commercial antibodies by western blot, and sixteen by immunoprecipitation, and immunofluorescence, comparing the signal produced by the antibodies in human A549 WT and TGM2 KO cells. Several high-quality antibodies that successfully detect TGM2 under our standardized experimental protocol can be identified. Researchers who wish to study TGM2 in a different species are encouraged to select high-quality antibodies, based on the results of this study, and investigate the predicted species reactivity of the manufacturer before extending their research. In our effort to address the antibody reliability and reproducibility challenges in scientific research, the authors recommend the antibodies that demonstrated to be underperforming under our standard procedure be removed from the commercial antibody market. Following the release of the antibody characterization, ab421 was removed from the manufacturer’s antibody catalog. 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. 27 The underlying data for this study can be found on Zenodo, an open-access repository for which YCharOS has its own collection of antibody characterization reports. 28 , 29 Methods The standardized protocols used to carry out this KO cell line-based antibody characterization platform was established and approved by a collaborative group of academics, industry researchers and antibody manufacturers. The detailed materials and step-by-step protocols used to characterize antibodies in western blot, immunoprecipitation and immunofluorescence are openly available on Protocol Exchange, a repository dedicated to openly sharing scientific research protocols, DOI: 10.21203/rs.3.pex-2607/v1 . 24 Antibodies and cell line used Cell lines used and primary antibodies tested in this study are listed in Table 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. 30 , 31 Table 2. Summary of the TGM2 antibodies tested. Company Catalog number Lot number RRID (Antibody Registry) Clonality Clone ID Host Immunogenic region Concentration (μg/μl) Vendors recommended applications Number of citations ( CiteAb.com ) Abcam ab109121** 1058833-3 AB_10861115 Recombinant-mono EPR2956 rabbit n/a 2.79 Wb 1 Abcam ab109200** 1044092-1 AB_10860177 Recombinant-mono EP2957 rabbit proprietary information 0.30 Wb 12 Abcam ab2386* 1051063-6 AB_2287299 Monoclonal CUB 7402 mouse proprietary information n/a Wb, IF 49 Abcam ab310333** 1056093-5 AB_3076417 Recombinant-mono EPR28142-86 rabbit proprietary information 0.50 Wb, IF 0 Abcam ab421 1034725-7 AB_304372 Polyclonal - rabbit proprietary information n/a Wb, IF 37 ABCD ABCD_AI748** 10/27/2023 AB_3076341 Recombinant-mono 679-14-E06 rabbit proprietary information 0.12 Others 0 ABclonal A21184** 3522042510 AB_3083448 Recombinant-mono ARC52843 rabbit aa 438-687 1.30 Wb, IF n/a Aviva Systems Biology ARP47471 QC18320-43546 AB_1107120 Polyclonal - rabbit middle region 0.50 Wb n/a Aviva Systems Biology ARP47472 QC16720 AB_1088480 Polyclonal - rabbit N-terminal 0.50 Wb n/a R&D Systems (a Bio-Techne brand) AF4376 CFGU0119031 AB_10890213 Polyclonal - sheep recombinant fragment (Ala2-Ala687) 0.20 Wb 4 R&D Systems (a Bio-Techne brand) MAB4376* CFNO0119031 AB_10971763 Monoclonal 716620 mouse recombinant fragment (Ala2-Ala687) 0.20 Wb 2 Cell Signaling Technology 3557** 3 AB_2202883 Recombinant-mono D11A6 rabbit proprietary information 0.02 Wb 41 GeneTex GTX111702 44524 AB_1952227 Polyclonal - rabbit middle region 1.05 Wb, IF 8 Proteintech 15100-1-AP 00081307 AB_2202885 Polyclonal - rabbit recombinant fragment (Ag7439) 0.60 Wb, IP, IF 21 Proteintech 68006-1-Ig* 10023724 AB_2918753 Monoclonal 2D4C11 mouse recombinant fragment (Ag7462) 1.00 Wb, IF n/a Thermo Fisher Scientific MA5-32819** YJ4089240 AB_2810095 Recombinant-mono JU30-02 rabbit aa 578-627 1.00 Wb 0 Thermo Fisher Scientific MA5-12739* ZA4176225 AB_10985077 Monoclonal CUB 7402 mouse Purified guinea pig liver TGase II 0.20 Wb, IP, IF 115 Data availability Underlying data Zenodo: Antibody Characterization Report for TGM2 (Protein-glutamine gamma-glutamyltransferase 2), https://doi.org/10.5281/zenodo.10819348 . 28 Zenodo: Dataset for the TGM2 (Protein-glutamine gamma-glutamyltransferase 2) antibody screening study, https://doi.org/10.5281/zenodo.10927535 . 29 Protocol Exchange: A consensus for antibody characterization platform, https://doi.org/10.21203/rs.3.pex-2607/v1 . 24 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 knockout 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. An earlier version of this of this article can be found on Zenodo (DOI: 10.5281/zenodo.10819348 ). References 1. Jeong EM, Lee KB, Kim GE, et al. : Competitive Binding of Magnesium to Calcium Binding Sites Reciprocally Regulates Transamidase and GTP Hydrolysis Activity of Transglutaminase 2. Int. J. Mol. Sci. 2020; 21 (3). PubMed Abstract | Publisher Full Text | Free Full Text 2. Kanchan K, Ergülen E, Király R, et al. : Identification of a specific one amino acid change in recombinant human transglutaminase 2 that regulates its activity and calcium sensitivity. Biochem. J. 2013; 455 (3): 261–272. 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PubMed Abstract | Publisher Full Text | Free Full Text Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 17 May 2024 ADD YOUR COMMENT Comment Author details Author details 1 Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Québec, H3A 2B4, Canada Riham Ayoubi Roles: Formal Analysis, Investigation, Methodology, Validation, Visualization Maryam Fotouhi Roles: Investigation, Validation, Visualization Charles Alende Roles: Investigation, Validation, Visualization Sara González Bolívar Roles: Investigation, Validation, Visualization Kathleen Southern Roles: Writing – Original Draft Preparation, Writing – Review & Editing Carl Laflamme Roles: Conceptualization, Formal Analysis, Funding Acquisition, Methodology, Resources, Validation, Writing – Review & Editing Competing interests For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - ABCD antibodies - Abcam - Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific. Grant information This work was supported by the Michael J. Fox Foundation for Parkinson’s Research (MJFF) (grant no. 18331). This work was also supported by a grant from the Canadian Institutes of Health Research Foundation (grant no. FDN154305), the Government of Canada through Genome Canada, Genome Quebec and Ontario Genomics (grant no. OGI-210). RA is supported by 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: 30 Jul 2024, 13:481 https://doi.org/10.12688/f1000research.150684.2 version 1 Published: 17 May 2024, 13:481 https://doi.org/10.12688/f1000research.150684.1 Copyright © 2024 Ayoubi R et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Ayoubi R, Fotouhi M, Alende C et al. A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved, 1 approved with reservations] . F1000Research 2024, 13 :481 ( https://doi.org/10.12688/f1000research.150684.2 ) 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 30 Jul 2024 Revised Views 0 Cite How to cite this report: Jean S. Reviewer Report For: A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved, 1 approved with reservations] . F1000Research 2024, 13 :481 ( https://doi.org/10.5256/f1000research.169623.r316340 ) The direct URL for this report is: https://f1000research.com/articles/13-481/v2#referee-response-316340 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 29 Aug 2024 Steve Jean , Department of Immunology and Cell Biology, Université de Sherbrooke, Sherbrooke, Canada Approved VIEWS 0 https://doi.org/10.5256/f1000research.169623.r316340 In this data note, the authors evaluated a panel of commercial human transglutaminase 2 (TGM2) antibodies in three typical applications: immunoblotting, immunoprecipitation, and immunofluorescence. The manuscript is clear, and the data is well described. I am impressed that all the ... Continue reading READ ALL In this data note, the authors evaluated a panel of commercial human transglutaminase 2 (TGM2) antibodies in three typical applications: immunoblotting, immunoprecipitation, and immunofluorescence. The manuscript is clear, and the data is well described. I am impressed that all the raw data can be easily accessed on the YCHAROS Zenodo website, which will ease evaluation by the community of the various antibodies tested. While this data note only refers to previously published protocols, the antibody report associated with this manuscript provides all the methodological information required to understand the data and to draw conclusions about the observed results. Previous comments from other reviewers were also well addressed or discussed adequately. Limitations of the standardized approaches are nicely discussed. I thus think that the manuscript is adequate for indexing. I only have a minor point that could further ease WB and IF data interpretation. It would be helpful to have the exposure time for the various WBs. This would provide another layer of information and ease the choice between two specific antibodies. Exposure time could be added at the bottom of each WB in Figures 1 and 2. Imaging parameters (or simply exposure time) could also be included in the legend of Figure 4. It is unclear if similar imaging parameters were used for all antibodies or if those were optimized for the best signal-to-noise ratio. Typo : In the legend of Figure 3 (page 6), please add ‘G’ to the end of the sentence ‘… pre-coupled to Dynabeads protein A or protein.‘ Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Membrane trafficking, RAB GTPases, phosphoinositides, Drosophila, proteomics, autophagy. 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 Jean S. Reviewer Report For: A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved, 1 approved with reservations] . F1000Research 2024, 13 :481 ( https://doi.org/10.5256/f1000research.169623.r316340 ) The direct URL for this report is: https://f1000research.com/articles/13-481/v2#referee-response-316340 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Bianchi N. Reviewer Report For: A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved, 1 approved with reservations] . F1000Research 2024, 13 :481 ( https://doi.org/10.5256/f1000research.169623.r308876 ) The direct URL for this report is: https://f1000research.com/articles/13-481/v2#referee-response-308876 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 21 Aug 2024 Nicoletta Bianchi , University of Ferrara, Ferrara, Italy Approved VIEWS 0 https://doi.org/10.5256/f1000research.169623.r308876 I have reviewed the changes made to the manuscript. I appreciate the inclusion of the number of times these antibodies have been reported in published articles, although the antibody panel has not been implemented. This still makes the widespread use ... Continue reading READ ALL I have reviewed the changes made to the manuscript. I appreciate the inclusion of the number of times these antibodies have been reported in published articles, although the antibody panel has not been implemented. This still makes the widespread use of some of them by the scientific community. However, note that the citations have been reported in Table 2 and not in Table 1 as you indicated. Minor revisions have been modified as requested, as well as a clear description of the preparation of lysates was included. For this reason, I approve the manuscript in this form. Best regards Nicoletta Bianchi Competing Interests: No competing interests were disclosed. Reviewer Expertise: cancer research 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 Bianchi N. Reviewer Report For: A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved, 1 approved with reservations] . F1000Research 2024, 13 :481 ( https://doi.org/10.5256/f1000research.169623.r308876 ) The direct URL for this report is: https://f1000research.com/articles/13-481/v2#referee-response-308876 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 17 May 2024 Views 0 Cite How to cite this report: Bianchi N. Reviewer Report For: A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved, 1 approved with reservations] . F1000Research 2024, 13 :481 ( https://doi.org/10.5256/f1000research.165276.r293962 ) The direct URL for this report is: https://f1000research.com/articles/13-481/v1#referee-response-293962 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 15 Jul 2024 Nicoletta Bianchi , University of Ferrara, Ferrara, Italy Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.165276.r293962 Dear Editorial Team, I find this work extremely interesting. The authors tested the efficacy and specificity of commercial antibodies that can be used to study TGM2. Unfortunately, only one cell line and its KO were used, this is ... Continue reading READ ALL Dear Editorial Team, I find this work extremely interesting. The authors tested the efficacy and specificity of commercial antibodies that can be used to study TGM2. Unfortunately, only one cell line and its KO were used, this is limiting, because the cell lines, especially cancer cells, can present different altered variants and/or different off-targets that could be recognized. At least the antibodies that are highly specific in this study could be tested on more cell lines. I believe that by contacting researchers who have developed TG2 KO lines or models, the authors could find a lot of willingness to collaborate. In any case, the manuscript is well done and of considerable applicative relevance and can help direct the researcher to a more appropriate choice of antibodies. However, for a better view of the applicability framework and before the indexing of this study, I suggest the inclusion of some antibodies from companies such as Zedira, which promotes a specific line of products for transglutaminase application, which are widely used by researchers working in the field of TGM2, as well as from Covalab. This would be of great interest and would greatly increase the impact of the work. Major revision: The authors should implement the panel of antibodies including those widely used in publications on TGM2. Minor revision: Keywords: typos errors, punctuation errors, “western” in capital letters, as well as when reported in the manuscript Introduction : “ The protein s activity is dependent on cellular location, remaining inactive intracellularly, and becoming activated upon secretion ”, the sentence is incorrect, because it is also active inside the cells depending on calcium release stimuli. “ by a committee of industry, academic research ”, which committee is this? must be specified. Some errors in the text. Results and discussion : Authors should clarify the difference between the cell extract and lysate used. If they mean the same thing, it would be better to use the same term. With my best regards, Nicoletta Bianchi 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: cancer research 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 Bianchi N. Reviewer Report For: A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved, 1 approved with reservations] . F1000Research 2024, 13 :481 ( https://doi.org/10.5256/f1000research.165276.r293962 ) The direct URL for this report is: https://f1000research.com/articles/13-481/v1#referee-response-293962 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 30 Jul 2024 Kathleen Southern , Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada 30 Jul 2024 Author Response Dear Nicoletta Bianchi, Thank you for reviewing the article “A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence” recently ... Continue reading Dear Nicoletta Bianchi, Thank you for reviewing the article “A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence” recently published to the YCharOS gateway. In response to your major and minor revisions ( italicized ), the authors have edited the existing manuscript and will be submitting an updated version of the article shortly. We hope our response to your review clarifies any concerns or misunderstandings you may have previously had. Reviewer Comment - Major revision: The authors should implement the panel of antibodies including those widely used in publications on TGM2. Author Response - To address your major revision, in the newly submitted manuscript, we have included a column to Table 1 to indicated the number of times the tested antibodies were cited in published articles. The information is provided on CiteAb.com . Please visit the website if you’d like to replicate this study using other widely used antibodies that were not evaluated in this study. Reviewer Comment - Minor revision Introduction : “The proteins activity is dependent on cellular location, remaining inactive intracellularly, and becoming activated upon secretion”, the sentence is incorrect, because it is also active inside the cells depending on calcium release stimuli. “by a committee of industry, academic research”, which committee is this? must be specified. Some errors in the text. Author Response - The sentence regarding the protein’s activity being associated to cellular location has been removed. As for the committee of industry and academic researchers, we have clearly indicated that the list of participating members is mentioned in the competing interest section Reviewer Comment - Results and discussion : Authors should clarify the difference between the cell extract and lysate used. If they mean the same thing, it would be better to use the same term. Author Response - In the second paragraph of this section, we have removed the word “extracts” to prevent misinterpretation and have clarified that cells were collected as lysates (medium free) or on culture medium to test the antibodies by western blot. Dear Nicoletta Bianchi, Thank you for reviewing the article “A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence” recently published to the YCharOS gateway. In response to your major and minor revisions ( italicized ), the authors have edited the existing manuscript and will be submitting an updated version of the article shortly. We hope our response to your review clarifies any concerns or misunderstandings you may have previously had. Reviewer Comment - Major revision: The authors should implement the panel of antibodies including those widely used in publications on TGM2. Author Response - To address your major revision, in the newly submitted manuscript, we have included a column to Table 1 to indicated the number of times the tested antibodies were cited in published articles. The information is provided on CiteAb.com . Please visit the website if you’d like to replicate this study using other widely used antibodies that were not evaluated in this study. Reviewer Comment - Minor revision Introduction : “The proteins activity is dependent on cellular location, remaining inactive intracellularly, and becoming activated upon secretion”, the sentence is incorrect, because it is also active inside the cells depending on calcium release stimuli. “by a committee of industry, academic research”, which committee is this? must be specified. Some errors in the text. Author Response - The sentence regarding the protein’s activity being associated to cellular location has been removed. As for the committee of industry and academic researchers, we have clearly indicated that the list of participating members is mentioned in the competing interest section Reviewer Comment - Results and discussion : Authors should clarify the difference between the cell extract and lysate used. If they mean the same thing, it would be better to use the same term. Author Response - In the second paragraph of this section, we have removed the word “extracts” to prevent misinterpretation and have clarified that cells were collected as lysates (medium free) or on culture medium to test the antibodies by western blot. Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 30 Jul 2024 Kathleen Southern , Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada 30 Jul 2024 Author Response Dear Nicoletta Bianchi, Thank you for reviewing the article “A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence” recently ... Continue reading Dear Nicoletta Bianchi, Thank you for reviewing the article “A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence” recently published to the YCharOS gateway. In response to your major and minor revisions ( italicized ), the authors have edited the existing manuscript and will be submitting an updated version of the article shortly. We hope our response to your review clarifies any concerns or misunderstandings you may have previously had. Reviewer Comment - Major revision: The authors should implement the panel of antibodies including those widely used in publications on TGM2. Author Response - To address your major revision, in the newly submitted manuscript, we have included a column to Table 1 to indicated the number of times the tested antibodies were cited in published articles. The information is provided on CiteAb.com . Please visit the website if you’d like to replicate this study using other widely used antibodies that were not evaluated in this study. Reviewer Comment - Minor revision Introduction : “The proteins activity is dependent on cellular location, remaining inactive intracellularly, and becoming activated upon secretion”, the sentence is incorrect, because it is also active inside the cells depending on calcium release stimuli. “by a committee of industry, academic research”, which committee is this? must be specified. Some errors in the text. Author Response - The sentence regarding the protein’s activity being associated to cellular location has been removed. As for the committee of industry and academic researchers, we have clearly indicated that the list of participating members is mentioned in the competing interest section Reviewer Comment - Results and discussion : Authors should clarify the difference between the cell extract and lysate used. If they mean the same thing, it would be better to use the same term. Author Response - In the second paragraph of this section, we have removed the word “extracts” to prevent misinterpretation and have clarified that cells were collected as lysates (medium free) or on culture medium to test the antibodies by western blot. Dear Nicoletta Bianchi, Thank you for reviewing the article “A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence” recently published to the YCharOS gateway. In response to your major and minor revisions ( italicized ), the authors have edited the existing manuscript and will be submitting an updated version of the article shortly. We hope our response to your review clarifies any concerns or misunderstandings you may have previously had. Reviewer Comment - Major revision: The authors should implement the panel of antibodies including those widely used in publications on TGM2. Author Response - To address your major revision, in the newly submitted manuscript, we have included a column to Table 1 to indicated the number of times the tested antibodies were cited in published articles. The information is provided on CiteAb.com . Please visit the website if you’d like to replicate this study using other widely used antibodies that were not evaluated in this study. Reviewer Comment - Minor revision Introduction : “The proteins activity is dependent on cellular location, remaining inactive intracellularly, and becoming activated upon secretion”, the sentence is incorrect, because it is also active inside the cells depending on calcium release stimuli. “by a committee of industry, academic research”, which committee is this? must be specified. Some errors in the text. Author Response - The sentence regarding the protein’s activity being associated to cellular location has been removed. As for the committee of industry and academic researchers, we have clearly indicated that the list of participating members is mentioned in the competing interest section Reviewer Comment - Results and discussion : Authors should clarify the difference between the cell extract and lysate used. If they mean the same thing, it would be better to use the same term. Author Response - In the second paragraph of this section, we have removed the word “extracts” to prevent misinterpretation and have clarified that cells were collected as lysates (medium free) or on culture medium to test the antibodies by western blot. Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Rieger MA. Reviewer Report For: A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved, 1 approved with reservations] . F1000Research 2024, 13 :481 ( https://doi.org/10.5256/f1000research.165276.r289998 ) The direct URL for this report is: https://f1000research.com/articles/13-481/v1#referee-response-289998 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 03 Jul 2024 Michael A Rieger , German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Goethe University, Cardio-Pulmonary-Institute, Frankfurt Cancer Institute, Frankfurt am Main, Germany; Hospital of the Goethe University Frankfurt Department of Medicine II Hematology Oncology Rheumatology Infectious Diseases HIV Therapy, Frankfurt, Hesse, Germany Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.165276.r289998 The authors of this study have tested 17 different commercially available antibodies against human Transglutaminase 2 (TGM2) on their specificity and feasibility for different biochemical and cell biological applications. They rigorously tested the antibodies for their suitability in Western blotting, ... Continue reading READ ALL The authors of this study have tested 17 different commercially available antibodies against human Transglutaminase 2 (TGM2) on their specificity and feasibility for different biochemical and cell biological applications. They rigorously tested the antibodies for their suitability in Western blotting, immunoprecipitation and immune fluorescence. TGM2 is a multifunctional enzyme found in the cytosol and in the extracellular matrix. It functions as a glutaminase and a GDPase, depending on its conformation governed by the presence of Ca2+, thereby catalyzing peptide cross-linking and conducting signal transduction. TGM2 has been implicated in several diseases, such as celiac disease, cancer, neurodegenerative diseases, among others. Therefore, TGM2 has served as potential target for drug interference strategies in ongoing preclinical and clinical trials. The work presented here is of utmost interest for anybody working on the physiological and pathological function of TGM2, and provides solid data for selecting appropriate reagents to specifically and robustly detect TGM2 for research and diagnostic purposes. In general, work performed by the authors should be a prerequisite before using commercially available reagents (especially antibodies) to study biological effects and draw potentially wide-ranging conclusions which solely depend on the chosen diagnostic tool. Therefore, the manuscript here serves as a blueprint of how to test the validity of commercially available antibodies for several commonly used analytical methods. The authors provide here a most valuable resource of data, based on appropriately planned and conducted experiments. The manuscript is well written with high quality data representation, and the described methods are sound. The results shown here are of qualitative nature, and do not allow direct comparison of binding kinetics and sensitivity of the tested antibodies. Clear differences in the specificity, accuracy and suitability of the selected antibodies for the tested methods can be demonstrated, which will have consequences for evaluating and selecting TGM2 antibodies for future applications and studies, and for critical re-evaluation of findings from published studies. Last, vendors will decide on future continuation and/or recommendation of their products, based on these results. A few important minor points are still missing in this report, which the authors may want to address: The sensitivity and specificity of antibodies depend on the correctly titrated amount and concentration used for the respective assay. The authors refer to their general protocol on how to test antibodies for their applications, but they do not provide an easy-to-see overview of the used concentrations of the presented TGM2 antibodies for each application. The authors may want to extend the information shown in Table 2 for each application presented in figures 1-4. TGM2 is expressed in different isoforms, and at least four isoforms have been robustly reported and studied. These isoforms differ in their c-terminal part of the protein, and only isoform 2 is significantly shorter with reduced molecular weight that can be seen by Western. It would be of interest for the research community to learn about the isoform specificity of the tested antibodies. The authors should at least provide a table with the immunogens/peptides used to generate these antibodies. In the long run, the consortium should consider to test isoform specificity by overexpressing TGM2 isoforms in null cell lines. Along the same lines, the authors should check for TGM2 mRNA expression of the different isoforms in their wt cell line A549 by isoform-specific qPCR. They should also briefly mention the different TGM2 isoforms and their functions in the introduction and point out the current limitations of their study regarding the isoform specificity. 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: Stem cells, Cancer stem cells, Single cell technologies, TGM2 physiology in colorectal cancer and other cancers 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 Rieger MA. Reviewer Report For: A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved, 1 approved with reservations] . F1000Research 2024, 13 :481 ( https://doi.org/10.5256/f1000research.165276.r289998 ) The direct URL for this report is: https://f1000research.com/articles/13-481/v1#referee-response-289998 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 18 Jul 2024 Kathleen Southern , Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada 18 Jul 2024 Author Response Dear Michael A. Rieger, The authors would like extend our gratitude for having taken the time to review this article and provide concrete feedback. Please see our response to your ... Continue reading Dear Michael A. Rieger, The authors would like extend our gratitude for having taken the time to review this article and provide concrete feedback. Please see our response to your points ( italicized ) which we have responded to and/or addressed in a new version of the article, which will be submitted to F1000 shortly. We hope to have clarified any concerns or misunderstandings you previously had and believe the article is now ready for approval. Reviewer Comment: 1. The sensitivity and specificity of antibodies depend on the correctly titrated amount and concentration used for the respective assay. The authors refer to their general protocol on how to test antibodies for their applications, but they do not provide an easy-to-see overview of the used concentrations of the presented TGM2 antibodies for each application. The authors may want to extend the information shown in Table 2 for each application presented in figures 1-4. Author Response: We understand how the concentration of antibodies used in each application were not clearly accessible in the report. Based on our in depth procedure referred to in methods section ( https://protocolexchange.researchsquare.com/article/pex-2607/v1) and the dilutions presented in the Figure legends, we can assume the viewers can determine which concentration was used for the respective assays or are able to reproduce the experiment based on the information provided. Reviewer Comment: 2. TGM2 is expressed in different isoforms, and at least four isoforms have been robustly reported and studied. These isoforms differ in their c-terminal part of the protein, and only isoform 2 is significantly shorter with reduced molecular weight that can be seen by Western. It would be of interest for the research community to learn about the isoform specificity of the tested antibodies. The authors should at least provide a table with the immunogens/peptides used to generate these antibodies. In the long run, the consortium should consider to test isoform specificity by overexpressing TGM2 isoforms in null cell lines. Author Response: We understand this need and have added a column to table 2 to provide the readers with the immunogenic or peptide region the antibodies target, based on the information provided on the antibody manufacturer catalog. Reviewer Comment: 3. Along the same lines, the authors should check for TGM2 mRNA expression of the different isoforms in their wt cell line A549 by isoform-specific qPCR. They should also briefly mention the different TGM2 isoforms and their functions in the introduction and point out the current limitations of their study regarding the isoform specificity. Author Response: The authors are aware of that TGM2 isoforms exists, but that being said the purpose of this Data note is not to provide information regarding those isoforms but rather deliver a toolkit, to be used by researchers who wish to study TGM2 in relation to health and/or disease, by demonstrating which commercially available antibodies are of high-quality to be selected and used to enhance their research. Using these antibodies, these researchers can dive deeper and decipher whether the isoforms are present in the cell line. This level of detail is outside the scope of this initiative, which follows a platform (see link to protocol exchange preprint above) to validate antibodies by creating/obtaining knockout (KO) cell lines for a protein target and screening all commercial antibodies against that target head-to-head in key academic applications, comparing wild-type to KO cells. Dear Michael A. Rieger, The authors would like extend our gratitude for having taken the time to review this article and provide concrete feedback. Please see our response to your points ( italicized ) which we have responded to and/or addressed in a new version of the article, which will be submitted to F1000 shortly. We hope to have clarified any concerns or misunderstandings you previously had and believe the article is now ready for approval. Reviewer Comment: 1. The sensitivity and specificity of antibodies depend on the correctly titrated amount and concentration used for the respective assay. The authors refer to their general protocol on how to test antibodies for their applications, but they do not provide an easy-to-see overview of the used concentrations of the presented TGM2 antibodies for each application. The authors may want to extend the information shown in Table 2 for each application presented in figures 1-4. Author Response: We understand how the concentration of antibodies used in each application were not clearly accessible in the report. Based on our in depth procedure referred to in methods section ( https://protocolexchange.researchsquare.com/article/pex-2607/v1) and the dilutions presented in the Figure legends, we can assume the viewers can determine which concentration was used for the respective assays or are able to reproduce the experiment based on the information provided. Reviewer Comment: 2. TGM2 is expressed in different isoforms, and at least four isoforms have been robustly reported and studied. These isoforms differ in their c-terminal part of the protein, and only isoform 2 is significantly shorter with reduced molecular weight that can be seen by Western. It would be of interest for the research community to learn about the isoform specificity of the tested antibodies. The authors should at least provide a table with the immunogens/peptides used to generate these antibodies. In the long run, the consortium should consider to test isoform specificity by overexpressing TGM2 isoforms in null cell lines. Author Response: We understand this need and have added a column to table 2 to provide the readers with the immunogenic or peptide region the antibodies target, based on the information provided on the antibody manufacturer catalog. Reviewer Comment: 3. Along the same lines, the authors should check for TGM2 mRNA expression of the different isoforms in their wt cell line A549 by isoform-specific qPCR. They should also briefly mention the different TGM2 isoforms and their functions in the introduction and point out the current limitations of their study regarding the isoform specificity. Author Response: The authors are aware of that TGM2 isoforms exists, but that being said the purpose of this Data note is not to provide information regarding those isoforms but rather deliver a toolkit, to be used by researchers who wish to study TGM2 in relation to health and/or disease, by demonstrating which commercially available antibodies are of high-quality to be selected and used to enhance their research. Using these antibodies, these researchers can dive deeper and decipher whether the isoforms are present in the cell line. This level of detail is outside the scope of this initiative, which follows a platform (see link to protocol exchange preprint above) to validate antibodies by creating/obtaining knockout (KO) cell lines for a protein target and screening all commercial antibodies against that target head-to-head in key academic applications, comparing wild-type to KO cells. Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 18 Jul 2024 Kathleen Southern , Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada 18 Jul 2024 Author Response Dear Michael A. Rieger, The authors would like extend our gratitude for having taken the time to review this article and provide concrete feedback. Please see our response to your ... Continue reading Dear Michael A. Rieger, The authors would like extend our gratitude for having taken the time to review this article and provide concrete feedback. Please see our response to your points ( italicized ) which we have responded to and/or addressed in a new version of the article, which will be submitted to F1000 shortly. We hope to have clarified any concerns or misunderstandings you previously had and believe the article is now ready for approval. Reviewer Comment: 1. The sensitivity and specificity of antibodies depend on the correctly titrated amount and concentration used for the respective assay. The authors refer to their general protocol on how to test antibodies for their applications, but they do not provide an easy-to-see overview of the used concentrations of the presented TGM2 antibodies for each application. The authors may want to extend the information shown in Table 2 for each application presented in figures 1-4. Author Response: We understand how the concentration of antibodies used in each application were not clearly accessible in the report. Based on our in depth procedure referred to in methods section ( https://protocolexchange.researchsquare.com/article/pex-2607/v1) and the dilutions presented in the Figure legends, we can assume the viewers can determine which concentration was used for the respective assays or are able to reproduce the experiment based on the information provided. Reviewer Comment: 2. TGM2 is expressed in different isoforms, and at least four isoforms have been robustly reported and studied. These isoforms differ in their c-terminal part of the protein, and only isoform 2 is significantly shorter with reduced molecular weight that can be seen by Western. It would be of interest for the research community to learn about the isoform specificity of the tested antibodies. The authors should at least provide a table with the immunogens/peptides used to generate these antibodies. In the long run, the consortium should consider to test isoform specificity by overexpressing TGM2 isoforms in null cell lines. Author Response: We understand this need and have added a column to table 2 to provide the readers with the immunogenic or peptide region the antibodies target, based on the information provided on the antibody manufacturer catalog. Reviewer Comment: 3. Along the same lines, the authors should check for TGM2 mRNA expression of the different isoforms in their wt cell line A549 by isoform-specific qPCR. They should also briefly mention the different TGM2 isoforms and their functions in the introduction and point out the current limitations of their study regarding the isoform specificity. Author Response: The authors are aware of that TGM2 isoforms exists, but that being said the purpose of this Data note is not to provide information regarding those isoforms but rather deliver a toolkit, to be used by researchers who wish to study TGM2 in relation to health and/or disease, by demonstrating which commercially available antibodies are of high-quality to be selected and used to enhance their research. Using these antibodies, these researchers can dive deeper and decipher whether the isoforms are present in the cell line. This level of detail is outside the scope of this initiative, which follows a platform (see link to protocol exchange preprint above) to validate antibodies by creating/obtaining knockout (KO) cell lines for a protein target and screening all commercial antibodies against that target head-to-head in key academic applications, comparing wild-type to KO cells. Dear Michael A. Rieger, The authors would like extend our gratitude for having taken the time to review this article and provide concrete feedback. Please see our response to your points ( italicized ) which we have responded to and/or addressed in a new version of the article, which will be submitted to F1000 shortly. We hope to have clarified any concerns or misunderstandings you previously had and believe the article is now ready for approval. Reviewer Comment: 1. The sensitivity and specificity of antibodies depend on the correctly titrated amount and concentration used for the respective assay. The authors refer to their general protocol on how to test antibodies for their applications, but they do not provide an easy-to-see overview of the used concentrations of the presented TGM2 antibodies for each application. The authors may want to extend the information shown in Table 2 for each application presented in figures 1-4. Author Response: We understand how the concentration of antibodies used in each application were not clearly accessible in the report. Based on our in depth procedure referred to in methods section ( https://protocolexchange.researchsquare.com/article/pex-2607/v1) and the dilutions presented in the Figure legends, we can assume the viewers can determine which concentration was used for the respective assays or are able to reproduce the experiment based on the information provided. Reviewer Comment: 2. TGM2 is expressed in different isoforms, and at least four isoforms have been robustly reported and studied. These isoforms differ in their c-terminal part of the protein, and only isoform 2 is significantly shorter with reduced molecular weight that can be seen by Western. It would be of interest for the research community to learn about the isoform specificity of the tested antibodies. The authors should at least provide a table with the immunogens/peptides used to generate these antibodies. In the long run, the consortium should consider to test isoform specificity by overexpressing TGM2 isoforms in null cell lines. Author Response: We understand this need and have added a column to table 2 to provide the readers with the immunogenic or peptide region the antibodies target, based on the information provided on the antibody manufacturer catalog. Reviewer Comment: 3. Along the same lines, the authors should check for TGM2 mRNA expression of the different isoforms in their wt cell line A549 by isoform-specific qPCR. They should also briefly mention the different TGM2 isoforms and their functions in the introduction and point out the current limitations of their study regarding the isoform specificity. Author Response: The authors are aware of that TGM2 isoforms exists, but that being said the purpose of this Data note is not to provide information regarding those isoforms but rather deliver a toolkit, to be used by researchers who wish to study TGM2 in relation to health and/or disease, by demonstrating which commercially available antibodies are of high-quality to be selected and used to enhance their research. Using these antibodies, these researchers can dive deeper and decipher whether the isoforms are present in the cell line. This level of detail is outside the scope of this initiative, which follows a platform (see link to protocol exchange preprint above) to validate antibodies by creating/obtaining knockout (KO) cell lines for a protein target and screening all commercial antibodies against that target head-to-head in key academic applications, comparing wild-type to KO cells. 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 17 May 2024 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 Version 2 (revision) 30 Jul 24 read read Version 1 17 May 24 read read Michael A Rieger , Frankfurt Cancer Institute, Frankfurt am Main, Germany; Hospital of the Goethe University Frankfurt Department of Medicine II Hematology Oncology Rheumatology Infectious Diseases HIV Therapy, Frankfurt, Germany Nicoletta Bianchi , University of Ferrara, Ferrara, Italy Steve Jean , Université de Sherbrooke, Sherbrooke, Canada 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 © 2024 Jean S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 29 Aug 2024 | for Version 2 Steve Jean , Department of Immunology and Cell Biology, Université de Sherbrooke, Sherbrooke, Canada 0 Views copyright © 2024 Jean S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions In this data note, the authors evaluated a panel of commercial human transglutaminase 2 (TGM2) antibodies in three typical applications: immunoblotting, immunoprecipitation, and immunofluorescence. The manuscript is clear, and the data is well described. I am impressed that all the raw data can be easily accessed on the YCHAROS Zenodo website, which will ease evaluation by the community of the various antibodies tested. While this data note only refers to previously published protocols, the antibody report associated with this manuscript provides all the methodological information required to understand the data and to draw conclusions about the observed results. Previous comments from other reviewers were also well addressed or discussed adequately. Limitations of the standardized approaches are nicely discussed. I thus think that the manuscript is adequate for indexing. I only have a minor point that could further ease WB and IF data interpretation. It would be helpful to have the exposure time for the various WBs. This would provide another layer of information and ease the choice between two specific antibodies. Exposure time could be added at the bottom of each WB in Figures 1 and 2. Imaging parameters (or simply exposure time) could also be included in the legend of Figure 4. It is unclear if similar imaging parameters were used for all antibodies or if those were optimized for the best signal-to-noise ratio. Typo : In the legend of Figure 3 (page 6), please add ‘G’ to the end of the sentence ‘… pre-coupled to Dynabeads protein A or protein.‘ Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Membrane trafficking, RAB GTPases, phosphoinositides, Drosophila, proteomics, autophagy. 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) Jean S. Peer Review Report For: A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved, 1 approved with reservations] . F1000Research 2024, 13 :481 ( https://doi.org/10.5256/f1000research.169623.r316340) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-481/v2#referee-response-316340 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 Bianchi N. 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. 21 Aug 2024 | for Version 2 Nicoletta Bianchi , University of Ferrara, Ferrara, Italy 0 Views copyright © 2024 Bianchi N. 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 I have reviewed the changes made to the manuscript. I appreciate the inclusion of the number of times these antibodies have been reported in published articles, although the antibody panel has not been implemented. This still makes the widespread use of some of them by the scientific community. However, note that the citations have been reported in Table 2 and not in Table 1 as you indicated. Minor revisions have been modified as requested, as well as a clear description of the preparation of lysates was included. For this reason, I approve the manuscript in this form. Best regards Nicoletta Bianchi Competing Interests No competing interests were disclosed. Reviewer Expertise cancer research 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) Bianchi N. Peer Review Report For: A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved, 1 approved with reservations] . F1000Research 2024, 13 :481 ( https://doi.org/10.5256/f1000research.169623.r308876) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-481/v2#referee-response-308876 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 Bianchi N. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 15 Jul 2024 | for Version 1 Nicoletta Bianchi , University of Ferrara, Ferrara, Italy 0 Views copyright © 2024 Bianchi N. 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 Dear Editorial Team, I find this work extremely interesting. The authors tested the efficacy and specificity of commercial antibodies that can be used to study TGM2. Unfortunately, only one cell line and its KO were used, this is limiting, because the cell lines, especially cancer cells, can present different altered variants and/or different off-targets that could be recognized. At least the antibodies that are highly specific in this study could be tested on more cell lines. I believe that by contacting researchers who have developed TG2 KO lines or models, the authors could find a lot of willingness to collaborate. In any case, the manuscript is well done and of considerable applicative relevance and can help direct the researcher to a more appropriate choice of antibodies. However, for a better view of the applicability framework and before the indexing of this study, I suggest the inclusion of some antibodies from companies such as Zedira, which promotes a specific line of products for transglutaminase application, which are widely used by researchers working in the field of TGM2, as well as from Covalab. This would be of great interest and would greatly increase the impact of the work. Major revision: The authors should implement the panel of antibodies including those widely used in publications on TGM2. Minor revision: Keywords: typos errors, punctuation errors, “western” in capital letters, as well as when reported in the manuscript Introduction : “ The protein s activity is dependent on cellular location, remaining inactive intracellularly, and becoming activated upon secretion ”, the sentence is incorrect, because it is also active inside the cells depending on calcium release stimuli. “ by a committee of industry, academic research ”, which committee is this? must be specified. Some errors in the text. Results and discussion : Authors should clarify the difference between the cell extract and lysate used. If they mean the same thing, it would be better to use the same term. With my best regards, Nicoletta Bianchi 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 cancer research 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 30 Jul 2024 Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada Dear Nicoletta Bianchi, Thank you for reviewing the article “A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence” recently published to the YCharOS gateway. In response to your major and minor revisions ( italicized ), the authors have edited the existing manuscript and will be submitting an updated version of the article shortly. We hope our response to your review clarifies any concerns or misunderstandings you may have previously had. Reviewer Comment - Major revision: The authors should implement the panel of antibodies including those widely used in publications on TGM2. Author Response - To address your major revision, in the newly submitted manuscript, we have included a column to Table 1 to indicated the number of times the tested antibodies were cited in published articles. The information is provided on CiteAb.com . Please visit the website if you’d like to replicate this study using other widely used antibodies that were not evaluated in this study. Reviewer Comment - Minor revision Introduction : “The proteins activity is dependent on cellular location, remaining inactive intracellularly, and becoming activated upon secretion”, the sentence is incorrect, because it is also active inside the cells depending on calcium release stimuli. “by a committee of industry, academic research”, which committee is this? must be specified. Some errors in the text. Author Response - The sentence regarding the protein’s activity being associated to cellular location has been removed. As for the committee of industry and academic researchers, we have clearly indicated that the list of participating members is mentioned in the competing interest section Reviewer Comment - Results and discussion : Authors should clarify the difference between the cell extract and lysate used. If they mean the same thing, it would be better to use the same term. Author Response - In the second paragraph of this section, we have removed the word “extracts” to prevent misinterpretation and have clarified that cells were collected as lysates (medium free) or on culture medium to test the antibodies by western blot. View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Bianchi N. Peer Review Report For: A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved, 1 approved with reservations] . F1000Research 2024, 13 :481 ( https://doi.org/10.5256/f1000research.165276.r293962) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-481/v1#referee-response-293962 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 Rieger 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. 03 Jul 2024 | for Version 1 Michael A Rieger , German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Goethe University, Cardio-Pulmonary-Institute, Frankfurt Cancer Institute, Frankfurt am Main, Germany; Hospital of the Goethe University Frankfurt Department of Medicine II Hematology Oncology Rheumatology Infectious Diseases HIV Therapy, Frankfurt, Hesse, Germany 0 Views copyright © 2024 Rieger 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 The authors of this study have tested 17 different commercially available antibodies against human Transglutaminase 2 (TGM2) on their specificity and feasibility for different biochemical and cell biological applications. They rigorously tested the antibodies for their suitability in Western blotting, immunoprecipitation and immune fluorescence. TGM2 is a multifunctional enzyme found in the cytosol and in the extracellular matrix. It functions as a glutaminase and a GDPase, depending on its conformation governed by the presence of Ca2+, thereby catalyzing peptide cross-linking and conducting signal transduction. TGM2 has been implicated in several diseases, such as celiac disease, cancer, neurodegenerative diseases, among others. Therefore, TGM2 has served as potential target for drug interference strategies in ongoing preclinical and clinical trials. The work presented here is of utmost interest for anybody working on the physiological and pathological function of TGM2, and provides solid data for selecting appropriate reagents to specifically and robustly detect TGM2 for research and diagnostic purposes. In general, work performed by the authors should be a prerequisite before using commercially available reagents (especially antibodies) to study biological effects and draw potentially wide-ranging conclusions which solely depend on the chosen diagnostic tool. Therefore, the manuscript here serves as a blueprint of how to test the validity of commercially available antibodies for several commonly used analytical methods. The authors provide here a most valuable resource of data, based on appropriately planned and conducted experiments. The manuscript is well written with high quality data representation, and the described methods are sound. The results shown here are of qualitative nature, and do not allow direct comparison of binding kinetics and sensitivity of the tested antibodies. Clear differences in the specificity, accuracy and suitability of the selected antibodies for the tested methods can be demonstrated, which will have consequences for evaluating and selecting TGM2 antibodies for future applications and studies, and for critical re-evaluation of findings from published studies. Last, vendors will decide on future continuation and/or recommendation of their products, based on these results. A few important minor points are still missing in this report, which the authors may want to address: The sensitivity and specificity of antibodies depend on the correctly titrated amount and concentration used for the respective assay. The authors refer to their general protocol on how to test antibodies for their applications, but they do not provide an easy-to-see overview of the used concentrations of the presented TGM2 antibodies for each application. The authors may want to extend the information shown in Table 2 for each application presented in figures 1-4. TGM2 is expressed in different isoforms, and at least four isoforms have been robustly reported and studied. These isoforms differ in their c-terminal part of the protein, and only isoform 2 is significantly shorter with reduced molecular weight that can be seen by Western. It would be of interest for the research community to learn about the isoform specificity of the tested antibodies. The authors should at least provide a table with the immunogens/peptides used to generate these antibodies. In the long run, the consortium should consider to test isoform specificity by overexpressing TGM2 isoforms in null cell lines. Along the same lines, the authors should check for TGM2 mRNA expression of the different isoforms in their wt cell line A549 by isoform-specific qPCR. They should also briefly mention the different TGM2 isoforms and their functions in the introduction and point out the current limitations of their study regarding the isoform specificity. 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 Stem cells, Cancer stem cells, Single cell technologies, TGM2 physiology in colorectal cancer and other cancers 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 18 Jul 2024 Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada Dear Michael A. Rieger, The authors would like extend our gratitude for having taken the time to review this article and provide concrete feedback. Please see our response to your points ( italicized ) which we have responded to and/or addressed in a new version of the article, which will be submitted to F1000 shortly. We hope to have clarified any concerns or misunderstandings you previously had and believe the article is now ready for approval. Reviewer Comment: 1. The sensitivity and specificity of antibodies depend on the correctly titrated amount and concentration used for the respective assay. The authors refer to their general protocol on how to test antibodies for their applications, but they do not provide an easy-to-see overview of the used concentrations of the presented TGM2 antibodies for each application. The authors may want to extend the information shown in Table 2 for each application presented in figures 1-4. Author Response: We understand how the concentration of antibodies used in each application were not clearly accessible in the report. Based on our in depth procedure referred to in methods section ( https://protocolexchange.researchsquare.com/article/pex-2607/v1) and the dilutions presented in the Figure legends, we can assume the viewers can determine which concentration was used for the respective assays or are able to reproduce the experiment based on the information provided. Reviewer Comment: 2. TGM2 is expressed in different isoforms, and at least four isoforms have been robustly reported and studied. These isoforms differ in their c-terminal part of the protein, and only isoform 2 is significantly shorter with reduced molecular weight that can be seen by Western. It would be of interest for the research community to learn about the isoform specificity of the tested antibodies. The authors should at least provide a table with the immunogens/peptides used to generate these antibodies. In the long run, the consortium should consider to test isoform specificity by overexpressing TGM2 isoforms in null cell lines. Author Response: We understand this need and have added a column to table 2 to provide the readers with the immunogenic or peptide region the antibodies target, based on the information provided on the antibody manufacturer catalog. Reviewer Comment: 3. Along the same lines, the authors should check for TGM2 mRNA expression of the different isoforms in their wt cell line A549 by isoform-specific qPCR. They should also briefly mention the different TGM2 isoforms and their functions in the introduction and point out the current limitations of their study regarding the isoform specificity. Author Response: The authors are aware of that TGM2 isoforms exists, but that being said the purpose of this Data note is not to provide information regarding those isoforms but rather deliver a toolkit, to be used by researchers who wish to study TGM2 in relation to health and/or disease, by demonstrating which commercially available antibodies are of high-quality to be selected and used to enhance their research. Using these antibodies, these researchers can dive deeper and decipher whether the isoforms are present in the cell line. This level of detail is outside the scope of this initiative, which follows a platform (see link to protocol exchange preprint above) to validate antibodies by creating/obtaining knockout (KO) cell lines for a protein target and screening all commercial antibodies against that target head-to-head in key academic applications, comparing wild-type to KO cells. View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Rieger MA. Peer Review Report For: A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved, 1 approved with reservations] . F1000Research 2024, 13 :481 ( https://doi.org/10.5256/f1000research.165276.r289998) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. 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