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). The proteins activity is dependent on cellular location, remaining inactive intracellularly, and becoming activated upon secretion. ^{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, academic research. 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

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).

For western blot experiments, we resolved proteins from WT and TGM2 KO cell extracts and probed them side-by-side with all antibodies in parallel on lysate and culture medium ( 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 immunoprecipitate ( 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. 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}

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