A guide to selecting high-performing antibodies for DJ-1 (PARK7) (Q99497) for use in western blot, immunoprecipitation, and immunofluorescence

Introduction

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 gateway⁵ and in Table 4 of this data note.⁴

Results and discussion

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).

Figure 1. DJ-1 antibody screening by western blot.

Lysates of HEK-293 T WT and PARK7 KO were prepared, and 30 μg of protein were processed for western blot with the indicated DJ-1 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 dilutions used: ab201147** at 1/5000; ab76008** at 1/5000; ab76241** at 1/2000; A0987 at 1/1000; ARP51267 at 1/500; MAB3995* at 1/1000; MAB39951* at 1/500; 5933** at 1/1000; GTX132552 at 1/2000; GTX132574 at 1/2000; GTX634830* at 1/2000; GTX634831* at 1/5000; MA5–32297** at 1/1000; MA5–33099* at 1/2000; MA5–35258** at 1/1000. Predicted band size: 20 kDa. * = monoclonal antibody, ** = recombinant antibody.

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).

Figure 2. DJ-1 antibody screening by immunoprecipitation.

HEK-293 T lysates were prepared, and immunoprecipitation was performed using 0.5 mg of lysate and 2.0 μg of the indicated DJ-1 antibodies pre-coupled to Dynabeads protein A or protein G. Samples were washed and processed for western blot with the DJ-1 antibody ab76008** used at 1/5000. The Ponceau stained transfers of each blot are shown. SM = 6% starting material; UB = 6% unbound fraction; IP = immunoprecipitate. * = monoclonal antibody, ** = recombinant antibody.

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

Figure 3. DJ-1 antibody screening by immunofluorescence.

HEK-293 T WT and PARK7 KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio on a 96-well collagen-coated plate with optically clear flat-bottom. Cells were stained with the indicated DJ-1 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (WT), red (antibody staining) and far-red (KO) channels was performed. Representative images of the blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, respectively. When an antibody was recommended for immunofluorescence by the supplier, we tested it at the recommended dilution. The rest of the antibodies were tested at 1 and 2 μg/ml, and the final concentration was selected based on the detection range of the microscope used and a quantitative analysis not shown here. Antibody dilutions used: ab201147** at 1/500; ab76008** at 1/100; ab76241** at 1/100; A0987 at 1/1500; ARP51267 at 1/200; MAB3995* at 1/500; MAB39951* at 1/500; 5933** at 1/50; GTX132552 at 1/100; GTX132574 at 1/500; GTX634830* at 1/1400; GTX634831* at 1/500; MA5–32297** at 1/1000; MA5–33099* at 1/100; MA5–35258** at 1/60. Bars = 10 μm. * = monoclonal antibody, ** = recombinant antibody.

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.

Limitations

Inherent limitations are associated with the antibody characterization platform used in this study. Firstly, the YCharOS project focuses on renewable (recombinant and monoclonal) antibodies and does not test all commercially available 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.

Method

The standardized protocols used to carry out this KO cell line-based antibody characterization platform was established and approved by a collaborative group of academics, industry researchers and antibody manufacturers. The detailed materials and step-by-step protocols used to characterize antibodies in western blot, immunoprecipitation and immunofluorescence are openly available on 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.

Cell lines and antibodies

The cell lines, primary and secondary antibodies used in this study are listed in Table 1, 2, and 3, respectively. To ensure consistency with manufacturer recommendations and account for proprietary formulations (where antibody concentrations are not disclosed), antibody usage is reported as dilution ratios rather than absolute concentrations. To facilitate proper citation and unambiguous identification, all cell lines and antibodies are referenced with their corresponding Research Resource Identifiers (RRIDs).^9,10 All cell lines used in this study were regularly tested for mycoplasma contamination and were confirmed to be mycoplasma-free.

Table 2. Summary of the DJ-1 antibodies tested.

Company	Catalog number	Lot number	RRID (Antibody Registry)	Clonality	Clone ID	Host	Concentration (μg/μL)	Vendors recommended applications
Abcam	ab201147**	1106441–1	AB_1310549	recombinant mono	EPR19466–105	rabbit	0.52	Wb, IP, IF
Abcam	ab76008**	1018054–21	AB_2283514	recombinant mono	EP2815Y	rabbit	0.11	Wb, IP, IF
Abcam	ab76241**	1097332–2	AB_2757506	recombinant mono	EP2816Y	rabbit	0.26	Wb
ABclonal	A0987	0001720101	AB_2047400	polyclonal	-	rabbit	1.74	Wb, IF
Aviva Systems Biology	ARP51267_P050	QC17282–100430	AB_2160100	polyclonal	-	rabbit	0.50	Wb
Bio-Techne (R&D Systems)	MAB3995*	CBLD0114061	AB_3658217	monoclonal	421015	rat	0.50	Wb
Bio-Techne (R&D Systems)	MAB39951*	CJMT0123101	AB_11179085	monoclonal	925805R	mouse	0.50	Wb
Cell Signaling Technology	5933**	2	AB_2886673	recombinant mono	D29E5	rabbit	0.13	Wb, IP, IF
GeneTex	GTX132552	42424	AB_2886682	polyclonal	-	rabbit	0.20	Wb
GeneTex	GTX132574	42508	AB_2888485	polyclonal	-	rabbit	0.22	Wb, IF
GeneTex	GTX634830*	43346	AB_2888486	monoclonal	GT353	mouse	1.40	Wb
GeneTex	GTX634831*	43346	AB_2809582	monoclonal	GT136	mouse	2.00	Wb, IF
Thermo Fisher Scientific	MA5–32297**	AC4649685	AB_2802649	recombinant mono	SN07–21	rabbit	1.00	Wb, IF
Thermo Fisher Scientific	MA5–33099*	AC4650511	AB_2849161	monoclonal	4H4	mouse	1.00	Wb, IF
Thermo Fisher Scientific	MA5–35258**	AC4650228	AB_1310549	recombinant mono	1Q7F8	rabbit	0.06	Wb

* = monoclonal antibody,

** = recombinant antibody

Table 3. Table of secondary antibodies used.

Company	Secondary antibody	Catalog number	RRID (Antibody Registry)	Clonality	Concentration (μg/μL)	Working concentration (μg/mL)
Proteintech	HRP-Goat Anti-Rabbit Antibody (H + L)	RGAR001	AB_3073505	recombinant polyclonal	1.0	0.05
Proteintech	HRP-Goat Anti-Mouse Antibody (H + L)	RGAM001	AB_3068333	recombinant polyclonal	1.0	0.5
Thermo Fisher Scientific	HRP-Goat Anti-Rat Antibody (H + L)	31470	AB_228356	polyclonal	0.8	0.4
Cell Signaling Technology	Protein A, HRP conjugate	12291	NA	polyclonal	0.125	0.5
Proteintech	CoraLite Plus 555-Goat Anti-Rabbit Antibody (H + L)	RGAR003	AB_3073507	recombinant polyclonal	0.5	0.5
Proteintech	CoraLite Plus 555-Goat Anti-Mouse Antibody (H + L)	RGAM003	AB_3068539	recombinant polyclonal	0.5	0.5
Invitrogen	Alexa Fluor™ 555-Goat anti-Rat IgG (H + L)	A-21434	AB_141733	polyclonal	2	0.5

Table 4. Illustrations to assess antibody performance in all western blot, immunoprecipitation and immunofluorescence.

Western blot	Immunoprecipitation	Immunofluorescence