Keywords
UniProt ID Q70CQ3, USP30, USP30, Ubiquitin-specific protease 30, Ubiquitin carboxyl-terminal hydrolase 30, Deubiquitinating enzyme 30, antibody characterization, antibody validation, western blot, immunoprecipitation, immunofluorescence
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Zha C, Ayoubi R, Alende C et al. A guide to selecting high-performing renewable antibodies for USP30 (Q70CQ3) across western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Research 2026, 15:1145 (https://doi.org/10.12688/f1000research.183778.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Data Note
A guide to selecting high-performing renewable antibodies for USP30 (Q70CQ3) across western blot, immunoprecipitation, and immunofluorescence
[version 1; peer review: awaiting peer review]
PUBLISHED 13 Jul 2026
OPEN PEER REVIEW
REVIEWER STATUS
This article is included in the YCharOS (Antibody Characterization through Open Science) gateway.
Abstract
Corresponding author:
Carl Laflamme
Competing interests:
For this project, the authors developed partnerships with leading antibody manufacturers and KO cell line
providers. The partners provide antibodies and KO cell lines to this project at no cost. These partners
include: Abbexa, Abcam, ABCD antibodies, ABclonal, Aviva Systems Biology, BioTechne, Cell Signaling
Technology, Developmental Studies Hybridoma Bank, GeneTex, Horizon Discovery (Revvity),
MilliporeSigma, Proteintech, 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 Quebec Consortium for Drug Discovery (CQDM), a grant from the Ministère de l’Économie, de l’Innovation et de l’Énergie du Québec.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Copyright:
© 2026 Zha C 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: Zha C, Ayoubi R, Alende C et al. A guide to selecting high-performing renewable antibodies for USP30 (Q70CQ3) across western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Research 2026, 15:1145 (https://doi.org/10.12688/f1000research.183778.1)
First published: 13 Jul 2026, 15:1145 (https://doi.org/10.12688/f1000research.183778.1)
Latest published: 13 Jul 2026, 15:1145 (https://doi.org/10.12688/f1000research.183778.1)
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Introduction
USP30 is a mitochondrial deubiquitinating enzyme that plays a pivotal role in the regulation of mitophagy, a selective autophagic process responsible for the removal of damaged mitochondria. Localized to the outer mitochondrial membrane, USP30 counteracts the activity of the PINK1/Parkin pathway by removing ubiquitin from mitochondrial substrates, thereby inhibiting mitochondrial clearance.1 This antagonistic function is essential for maintaining mitochondrial quality control and cellular homeostasis. Dysregulation of USP30 has been associated with the pathogenesis of several neurodegenerative disorders, particularly Parkinson’s disease, where impaired mitophagy contributes to the accumulation of dysfunctional mitochondria and neuronal loss.2
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.3 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.3 Here we characterized seven commercial USP30 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 USP30 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 gateway4 and in Table 5 of this data note.3
Results and discussion
Our standard protocol involves comparing readouts from wild type (WT) and KO cells.5,6 The first step was to identify a cell line(s) that expresses sufficient levels of a given protein to generate a measurable signal using antibodies. To this end, we examined the DepMap (Cancer Dependency Map Portal, RRID:SCR_017655) transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log2 (transcripts per million “TPM” + 1), which we have found to be a suitable cut-off.7 The cell lines HAP1 and MCF-7 express the USP30 transcript at 3.6 and 2.9 log2 TPM + 1, respectively. The HAP1 USP30 KO cell line was obtained from Horizon Discovery and the MCF-7 USP30 KO was from Abcam ( Table 1). Moreover, as seen on DepMap, the HAP1 and MCF-7 cell lines does not harbor mutations in the USP30 gene that could affect antibody–epitope binding.
Table 1. Summary of the cell lines used.
| Institution | Catalog number | RRID (Cellosaurus) | Cell line | Genotype |
|---|---|---|---|---|
| Horizon Discovery | C631 | CVCL_Y019 | HAP1 | WT |
| Horizon discovery | HZGHC007482c012 | CVCL_E2NN | HAP1 | USP30 KO |
| Abcam | ab271144 | CVCL_0031 | MCF-7 | WT |
| Abcam | ab324541 | - | MCF-7 | USP30 KO |
Two recombinant antibodies were first used to confirm protein expression by western blot in both KO lines ( Figure 1A). All seven antibodies were then screened by western blot using MCF-7 WT and USP30 KO protein lysates resolved on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with the seven USP30 antibodies in parallel ( Figure 1B).
Figure 1. USP30 antibody screening by western blot.
A) 30 μg of lysates from HAP1 and MCF-7 (WT and USP30 KO) were processed for western blot with the USP30 antibodies ab314749** and 57045** used at 1/1000. B) 30 μg of lysates from MCF-7 WT and USP30 KO were processed for western blot with the indicated USP30 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. Predicted band size: 58.5 kDa. ** = recombinant antibody, * = monoclonal antibody.
We then assessed the capability of all seven antibodies to capture USP30 from MCF-7 WT protein extracts using immunoprecipitation techniques, followed by western blot analysis. For the immunoblot step, a specific USP30 antibody, 83240–5-RR, 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. USP30 antibody screening by immunoprecipitation.
MCF-7 lysates were prepared, and immunoprecipitation was performed for 1 h using 0.5 mg of lysate and 2.0 μg of the indicated USP30 antibodies pre-coupled to Dynabeads protein A or protein G. Samples were washed and processed for western blot with the USP30 antibody 83240–5-RR** used at 1/1000. The Ponceau stained transfers of each blot are shown. SM = 6% starting material; UB = 6% unbound fraction; IP = immunoprecipitated. ** = recombinant antibody, * = monoclonal antibody.
For immunofluorescence, seven antibodies were screened using a mosaic strategy. First, MCF-7 WT and USP30 KO cells were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the USP30 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.3
Figure 3. USP30 antibody screening by immunofluorescence.
MCF-7 WT and USP30 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 in a 96-well plate with optically clear flat-bottom. Culture medium was removed, and cells were fixed with 4% PFA for 10 min at room temperature. Cells were permeabilized with 0.1% Triton-X100 or 0.05% Saponin for 10 min at room temperature and stained with the indicated USP30 antibodies and with the corresponding Fluor 555 coupled secondary antibody then with 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. WT/KO ratios are presented with each corresponding image. Bars = 10 μm. ** = recombinant antibody, * = monoclonal antibody.
In conclusion, we have screened seven USP30 commercial antibodies by western blot, immunoprecipitation, and immunofluorescence by comparing the signal produced by the antibodies in human MCF-7 WT and USP30 KO cells. To assist users in interpreting antibody performance, Table 5 outlines various scenarios in which antibodies may perform in all three applications.7 High-quality and renewable antibodies that successfully detect USP30 were identified in all applications. Researchers who wish to study USP30 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 USP30 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 USP30, only a brief overview of the protein’s function and its relevance in disease is provided. USP30 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 ).3 Brief descriptions of the experimental setup used to carry out this study can be found below.
Cell lines
The cell lines used in this study are listed in Table 1. To facilitate proper citation and unambiguous identification, all cell lines are referenced with their corresponding Research Resource Identifiers (RRIDs).8,9 All cell lines used in this study were regularly tested for mycoplasma contamination and were confirmed to be mycoplasma-free.
Primary and secondary antibodies
The primary antibodies are listed in Table 2 and referenced with their RRIDs. 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. The used antibody dilutions in western blot and immunofluorescence are listed in Table 3. In western blot, dilutions were chosen according to the recommendations of the antibody supplier and titrated when needed. When an antibody was recommended for immunofluorescence by the supplier, we tested it at the recommended dilution and at 1 or 2 μg/ml. The rest of the antibodies were tested at 1 and 2 μg/ml, and the final dilution was selected based on the detection range of the microscope used and a quantitative analysis not shown here. A standardized 2 μg of antibody is tested in immunoprecipitation. Volumes corresponding to 2 μg of each antibody are listed in Table 3. Secondary antibodies and their corresponding concentrations are listed in Table 4.
Table 2. Summary of the USP30 antibodies tested.
| Company | Catalog number | Lot number | RRID (Antibody Registry) | Clonality | Clone ID | Host | Concentration (μg/μL) | Vendors recommended applications |
|---|---|---|---|---|---|---|---|---|
| Abbexa | abx422618** | A2511755X | AB_3739797 | recombinant mono | U786 | rabbit | 1.00 | Wb |
| Abcam | ab314749** | 1068225-15 | AB_3739894 | recombinant mono | EPR27024–81 | rabbit | 0.50 | Wb |
| Aviva Systems Biology | ARP50098_P050 | QC29807–42530 | AB_10713144 | polyclonal | - | rabbit | 0.50 | Wb |
| Cell Signaling Technology | 57045** | 1 | AB_3739933 | recombinant mono | F3S4O | rabbit | 0.20 | Wb, IP, IF |
| Proteintech | 15402–1-AP | 00128697 | AB_3085460 | polyclonal | - | rabbit | 0.90 | Wb, IF |
| Proteintech | 83240–5-RR** | 23008073 | AB_3670916 | recombinant mono | 240111C6 | rabbit | 1.00 | Wb |
| Thermo Fisher Scientific | MA5–31401* | 79532352 | AB_2787037 | monoclonal | CL4438 | mouse | 1.00 | other |
Table 3. Dilutions of USP30 antibodies used in all western blot, immunoprecipitation and immunofluorescence.
Table 4. 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 |
| 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 |
Antibody screening by western blot
HAP1 and MCF-7 (WT and USP30 KO) cells were collected in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Thermo Fisher Scientific, cat. Number 89901) supplemented with 1x protease inhibitor cocktail mix (MilliporeSigma, cat. Number P8340). Lysates were sonicated briefly and incubated 30 min on ice. Lysates were spun at ~110,000 x g for 15 min at 4 °C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and western blot. BLUelf prestained protein ladder (GeneDireX, cat. Number PM008–0500) was used.
Western blots were performed with precast midi 4–20% Tris-Glycine polyacrylamide gels (Thermo Fisher Scientific, cat. Number WXP42012BOX) ran with Tris/Glycine/SDS buffer (Bio-Rad, cat. Number 1610772), loaded in Laemmli loading sample buffer (Thermo Fisher Scientific, cat. Number AAJ61337AD) and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau S staining (Thermo Fisher Scientific, cat. Number BP103–10) which is scanned to show together with individual western blot. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated O/N at 4 °C with 5% milk in TBS with 0,1% Tween 20 (TBST) (Cell Signalling Technology, cat. Number 9997). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated with the membrane in TBST with 5% milk for 1 hr at room temperature followed by three washes with TBST. Membranes were incubated with Pierce ECL (Thermo Fisher Scientific, cat. Number 32106) prior to detection with the iBright™ CL1500 Imaging System (Thermo Fisher Scientific, cat. Number A44240).
Antibody screening by immunoprecipitation
Antibody-bead conjugates were prepared by adding 2 μg to 500 μl of Pierce IP Lysis Buffer from Thermo Fisher Scientific (cat. Number 87788) in a microcentrifuge tube, together with 30 μl of Dynabeads protein A- (for rabbit antibodies) or protein G- (for mouse antibodies) (Thermo Fisher Scientific, cat. Number 10002D and 10004D, respectively). All tubes were rocked for ~1 h at 4 °C followed by two washes to remove unbound antibodies.
MCF-7 WT cells were collected in Pierce IP buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) supplemented with protease inhibitor. Lysates were rocked 30 min at 4 °C and spun at 110,000 x g for 15 min at 4 °C. 0.5 ml aliquots at 1 mg/ml of lysate were incubated with an antibody-bead conjugate for ~1 h at 4 °C. The unbound fractions were collected, and beads were subsequently washed three times with 1.0 ml of IP buffer and processed for SDS-PAGE and western blot on precast midi 4–20% Tris-Glycine polyacrylamide gels.
Antibody screening by immunofluorescence
MCF-7 WT and USP30 KO cells were labelled with a green and a far-red fluorescence dye, respectively (Thermo Fisher Scientific, cat. Number C2925 and C34565). WT and KO cells were plated in a 96-well plate with optically clear flat-bottom (Perkin Elmer, cat. Number 6055300) as a mosaic and incubated for 24 hrs in a cell culture incubator at 37 °C, 5% CO2. Culture medium was removed, and cells were fixed in 4% paraformaldehyde (PFA) (VWR, cat. Number 100503–917) in phosphate buffered saline (PBS) (Wisent, cat. Number 311–010-CL) for 10 min at room temperature. Cells were permeabilized in PBS1x with 0.05% Saponin (MilliporeSigma, cat. Number 47036) or 0.1% Triton X-100 (Thermo Fisher Scientific, cat. Number BP151–500) for 10 min at room temperature. Cells were blocked in IF buffer (PBS1x, 5% BSA, 0.005% Saponin or 0.01% Triton X-100) with 5% normal goat (Gibco, cat. Number 16210–064) serum for 1 hr at room temperature. Cells were incubated with the corresponding IF buffer containing the primary USP30 antibodies overnight at 4 °C. Cells were then washed 3 × 10 min with IF buffer and incubated with the corresponding Coralite plus 555-conjugated secondary antibody in IF buffer for 1 hr at room temperature. Cells were washed 3 × 10 min with PBS1x then incubated with DAPI (Thermo Fisher Scientific, cat. Number D3571) and washed once with PBS.
Images were acquired on an ImageXpress micro confocal high-content microscopy system (Molecular Devices), using a using a 20x NA 0.94 air objective and scientific CMOS cameras, equipped with 395, 475, 555 and 635 nm solid state LED lights (lumencor Aura III light engine) and bandpass filters to excite DAPI, Cellmask Green, Alexa-555 and Cellmask Red, respectively. Images had pixel sizes of 0.68 x 0.68 microns, and a z-interval of 4 microns. For analysis and visualization, shading correction (shade only) was carried out for all images. Then, maximum intensity projections were generated using 3 z-slices. Segmentation was carried out separately on maximum intensity projections of Cellmask channels using CellPose 1.0, and masks were used to generate outlines and for intensity quantification.10 Figures were assembled with Adobe Illustrator.
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how to cite this article
Zha C, Ayoubi R, Alende C et al. A guide to selecting high-performing renewable antibodies for USP30 (Q70CQ3) across western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Research 2026, 15:1145 (https://doi.org/10.12688/f1000research.183778.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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