DJ-1 is a highly conserved, redox-sensitive protein that plays a role in maintaining cellular homeostasis, regulating oxidative stress and preserving mitochondrial integrity. ¹ In neurons, DJ-1 stabilizes mitochondria by reducing oxidative stress and promoting the clearance of dysfunctional mitochondria through mitophagy, thereby preventing the accumulation of toxic reactive oxygen species (ROS) and mitigating neuronal damage. ² Beyond its protective role against oxidative damage, DJ-1 has been implicated in diverse cellular processes, including regulation of transcription factors, protection from protein aggregation, modulation of apoptosis, and maintenance of mitochondrial dynamics. Mutations in the PARK7 gene, which cause loss of function, are linked to early-onset familial Parkinson’s disease (PD) by predisposing dopaminergic neurons to degeneration. ³

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. ⁴ It consists of identifying human cell lines with adequate target protein expression and the development/contribution of equivalent knockout (KO) cell lines, followed by antibody characterization procedures using most commercially available renewable antibodies against the corresponding protein. ⁴ Here we characterized sixteen commercial DJ-1 antibodies, selected and donated by participant antibody manufacturers, for use in western blot, immunoprecipitation, and immunofluorescence (also referred to as immunocytochemistry), enabling biochemical and cellular assessment of DJ-1 properties and function.

The authors do not engage in result analysis or offer explicit antibody recommendations. 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 ⁵ and in Table 4 of this data note. ⁴

Our standard protocol involves comparing readouts from wild type (WT) and KO cells. ^{6, 7} The first step is to identify a cell line that expresses the target protein at sufficient levels to generate a measurable signal. Using the DepMap transcriptomics database (Cancer Dependency Map Portal, RRID: SCR_017655), we found that DJ-1 is highly and ubiquitously expressed across cancer cell lines. Accordingly, a PARK7 KO HEK-293 T cell line was obtained from Abcam ( Table 1).

To screen all sixteen antibodies by western blot, WT and PARK7 KO protein lysates were ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with sixteen DJ-1 antibodies in parallel ( Figure 1).

We then assessed the capability of all sixteen antibodies to capture DJ-1 from HEK-293 T protein extracts using immunoprecipitation techniques, followed by western blot analysis. For the immunoblot step, a specific DJ-1 antibody identified previously (refer to Figure 1) was selected. Equal amounts of the starting material (SM) and the unbound fractions (UB), as well as the whole immunoprecipitate (IP) eluates were separated by SDS-PAGE ( Figure 2).

For immunofluorescence, the sixteen DJ-1 antibodies were screened using a mosaic strategy. First, HEK-293 T WT and PARK7 KO cells were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the DJ-1 antibodies were evaluated. Both WT and KO lines imaged in the same field of view to reduce staining, imaging and image analysis bias ( Figure 3). Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested, and the images presented in Figure 3 are representative of this analysis. ⁴

In conclusion, we have screened sixteen DJ-1 commercial antibodies by western blot, immunoprecipitation, and immunofluorescence by comparing the signal produced by the antibodies in human HEK-293 T WT and PARK7 KO cells. To assist users in interpreting antibody performanyce, Table 4 outlines various scenarios in which antibodies may perform in all three applications. ⁸ High-quality and renewable antibodies that successfully detect DJ-1 antibodies were identified. Researchers who wish to study DJ-1 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.

Inherent limitations are associated with the antibody characterization platform used in this study. Firstly, the YCharOS project focuses on renewable (recombinant and monoclonal) antibodies and does not test all commercially available DJ-1 antibodies. YCharOS partners provide approximately 80% of all renewable antibodies, but some top-cited polyclonal antibodies may not be available through these partners. We encourage readers to consult vendor documentation to identify the specific antigen each antibody is raised against, where such information is available.

Secondly, the YCharOS effort employs a non-biased approach that is agnostic to the protein for which antibodies have been characterized. The aim is to provide objective data on antibody performance without preconceived notions about how antibodies should perform or the molecular weight that should be observed in western blot. As the authors are not experts in DJ-1, only a brief overview of the protein’s function and its relevance in disease is provided. DJ-1 experts are invited to analyze and interpret observed banding patterns in western blots and subcellular localization in immunofluorescence.

Thirdly, YCharOS experiments are not performed in replicates primarily due to the use of multiple antibodies targeting various epitopes. Once a specific antibody is identified, it validates the protein expression of the intended target in the selected cell line, confirms the lack of protein expression in the KO cell line and supports conclusions regarding the specificity of the other antibodies. All experiments are performed using master mixes, and meticulous attention is paid to sample preparation and experimental execution. In IF, the use of two different concentrations serves to evaluate antibody specificity and can aid in assessing assay reliability. In instances where antibodies yield no signal, a repeat experiment is conducted following titration. Additionally, our independent data is performed subsequently to the antibody manufacturers internal validation process, therefore making our characterization process a repeat.

Lastly, as comprehensive and standardized procedures are respected, any conclusions remain confined to the experimental conditions and cell line used for this study. The use of a single cell type for evaluating antibody performance poses as a limitation, as factors such as target protein abundance significantly impact results. Additionally, the use of cancer cell lines containing gene mutations poses a potential challenge, as these mutations may be within the epitope coding sequence or other regions of the gene responsible for the intended target. Such alterations can impact the binding affinity of antibodies. This represents an inherent limitation of any approach that employs cancer cell lines.

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 Protocols.io ( protocols.io/view/a-consensus-platform-for-antibody-characterization ). ⁴ Brief descriptions of the experimental setup used to carry out this study can be found below.