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

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.


Introduction
2][3] Encoded by TGM2 gene, TGM2 protein exhibits its function through Ca 2+ -dependent cross-linking of substrates, thereby modulating their activity. 1TGM2's catalytic activity is dependent on guanine nucleotides and Ca 2+ binding. 4Both GTP and/or GDP act as negative regulators of TGM2, inducing a conformational change upon binding, inhibiting its cross-linking activity (closed form).1][12][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. 14,156][17][18] Regulating TGM2 activity using inhibitors could positively affect the human diseases in which TGM2 is implicated. 19,20Identifying high-quality antibodies would accelerate TGM2 research and its potential as a pharmacological target.2][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. 25,26The 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).

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.
Any further responses from the reviewers can be found at the end of the article 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).
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.
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.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. 27e 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.

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. 24tibodies 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

Steve Jean
Department of Immunology and Cell Biology, Université de Sherbrooke, Sherbrooke, Canada 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.'

Are the protocols appropriate and is the work technically sound? Yes
Are sufficient details of methods and materials provided to allow replication by others?

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.
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.

Nicoletta Bianchi
University of Ferrara, Ferrara, Italy 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
Is the rationale for creating the dataset(s) clearly described?

Nicoletta Bianchi
University of Ferrara, Ferrara, Italy 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 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.
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 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.

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.The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com

Figure 3 .
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.

Reviewer Report 21
August 2024 https://doi.org/10.5256/f1000research.169623.r308876© 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.

3 .
Is the rationale for creating the dataset(s) clearly described?YesAre the protocols appropriate and is the work technically sound?YesAre sufficient details of methods and materials provided to allow replication by others?PartlyAre the datasets clearly presented in a useable and accessible format?YesCompeting Interests: No competing interests were disclosed.Reviewer Expertise: Stem cells, Cancer stem cells, Single cell technologies, TGM2 physiology in colorectal cancer and other cancersI 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.

Table 1 .
Summary of the cell lines used. 28,29

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.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Version 1
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.