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. 2024 Dec 20;13:1545. [Version 1] doi: 10.12688/f1000research.160121.1

A guide to selecting high-performing antibodies for ADNP (UniProt ID: Q9H2P0) for use in western blot, immunoprecipitation, and immunofluorescence

Vera Ruíz Moleón 1, Charles Alende 1, Maryam Fotouhi 1, Riham Ayoubi 1, Sara González Bolívar 1, Carl Laflamme 1,a; NeuroSGC/YCharOS/EDDU collaborative group; ABIF consortium
PMCID: PMC11822250  PMID: 39949964

Abstract

ADNP is a multifunctional protein involved in chromatin remodeling, transcription, and microtubule interaction, playing a critical role in brain development, with mutations linked to ADNP-Related Disorder. Here we have characterized seven ADNP commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are 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 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.

Keywords: Q9H2P0, ADNP, Activity-Dependent Neuroprotective Protein, Activity-Dependent Neuroprotector homeobox Protein, antibody characterization, antibody validation, western blot, immunoprecipitation, immunofluorescence

Introduction

ADNP (Activity-Dependent Neuroprotector Homeobox Protein) is part of the SWI/SNF chromatin remodeling complex, a key regulator of RNA transcription and splicing. 1 It is a multifunctional protein characterized by nine Zinc fingers, a nuclear localization signal and a DNA-binding homeobox domain, 2 which collectively support its role as a transcription factor. 3 The protein also contains a small NAP motif (amino acids “NAPVSIPQ”) site that contributes to its interaction with microtubules. 4 Given its function in brain development 5 and other roles in the cell, 6 ADNP deletion is lethal in mice and haploinsufficiency results in cognitive impairment. 7 Mutations in the ADNP gene are associated with several congenital abnormalities and intellectual disabilities, including Helsmoortel-Van der Aa Syndrome 8 and ADNP-related Autism Spectrum Disorder, 9 collectively termed “ ADNP-Related Disorder”. 10

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. 11– 13 Here we evaluated the performance of seven commercial antibodies for ADNP for use in western blot, immunoprecipitation, and immunofluorescence, enabling biochemical and cellular assessment of ADNP properties and function. The platform for antibody characterization used to carry out this study was endorsed by a committee of industry and academic representatives. 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 antibodies against the corresponding protein. The standardized consensus antibody characterization protocols are openly available on Protocol Exchange, a preprint server (DOI: 10.21203/rs.3.pex-2607/v1). 14

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. 15

Results and discussion

Our standard protocol involves comparing readouts from wild type (WT) and KO cell lines. 14, 16 In the absence of commercially available KO cell lines, siRNA technology can be employed to knockdown (KD) the target gene. 17, 18 To determine which cell line demonstrates high expression of ADNP and thus be appropriate for KD, the first step is to identify a cell line 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 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. 11 As a result, DMS 53 which expresses the ADNP transcript at 5.7 was identified as a suitable cell line to use here. A non-targeting siRNA pool was used to treat the DMS 53 control (ctrl) cells, while ADNP was KD using a pool of siRNA targeting this gene.

To screen all seven antibodies by western blot, ctrl and ADNP KD protein lysates were ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with the ADNP antibodies in parallel ( Figure 1).

Figure 1. ADNP antibody screening by western blot.

Figure 1.

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Lysates of DMS 53 ctrl and ADNP KD were prepared, and 30 μg of protein were processed for western blot with the indicated ADNP antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of ctrl and KD 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 dilution used: ab300114** at 1/1000, A4546 at 1/1000, NBP1-89236 at 1/250 (0.4 μg/ml), NBP2-97747 at 1/1000, 17987-1-AP at 1/1000, 702911** at 1/1000, PA5-52286 at 1/250 (0.4 μg/ml). Predicted band size: predicted band size 123.5 kDa. **Recombinant antibody.

We then assessed the capability of all seven antibodies to capture ADNP from DMS 53 protein extracts using immunoprecipitation techniques, followed by western blot analysis. For the immunoblot step, a specific ADNP 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. ADNP antibody screening by immunoprecipitation.

Figure 2.

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DMS 53 WT lysates were prepared, and immunoprecipitation was performed using 1 mg of lysate and 2.0 μg of the indicated ADNP antibodies pre-coupled to Dynabeads protein A or protein G. Samples were washed and processed for western blot with the indicated ADNP antibody. For western blot, 702911** was used at 1/500. The Ponceau stained transfers of each blot are shown. SM=6% starting material; UB=6% unbound fraction; IP=immunoprecipitate, **Recombinant antibody.

For immunofluorescence, the seven antibodies were screened using a mosaic strategy. First, DMS 53 ctrl and ADNP KD cells were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the ADNP antibodies were evaluated. Both ctrl and KD lines were imaged in the same field of view to reduce staining, imaging and image analysis bias ( Figure 3). Quantification of immunofluorescence intensity in hundreds of ctrl and KD cells was performed for each antibody tested, and the images presented in Figure 3 are representative of this analysis. 14

Figure 3. ADNP antibody screening by immunofluorescence.

Figure 3.

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DMS 53 ctrl and ADNP KD cells were labelled with a green or a far-red fluorescent dye, respectively. Ctrl and KD cells were mixed and plated to a 1:1 ratio on coverslips. Cells were stained with the indicated ADNP antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (ctrl), red (antibody staining) and far-red (KD) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. Ctrl and KD cells are outlined with green and magenta dashed lines, 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 dilution used: ab300114** at 1/500, A4546 at 1/700, NBP1-89236 at 1/100, NBP2-97747 at 1/300, 17987-1-AP at 1/600, 702911** at 1/500, PA5-52286 at 1/100. Bars = 10 μm. **Recombinant antibody.

In conclusion, we have screened seven ADNP commercial antibodies by western blot, immunoprecipitation, and immunofluorescence by comparing the signal produced by the antibodies in human DMS 53 ctrl and ADNP KD cells. To assist users in interpreting antibody performanyce, Table 3 outlines various scenarios in which antibodies may perform in all three applications. 11 Several high-quality and renewable antibodies that successfully detect ADNP were identified in all applications. Researchers who wish to study ADNP 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.

Table 3. Illustrations to assess antibody performance in all western blot, immunoprecipitation and immunofluorescence.

Western blot Immunoprecipitation Immunofluorescence
graphic file with name f1000research-13-175953-g0003.jpg graphic file with name f1000research-13-175953-g0004.jpg graphic file with name f1000research-13-175953-g0005.jpg
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This table has been reproduced with permission from Ayoubi et al., Elife, 2023. 11

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 ADNP antibodies. YCharOS partners provide approximately 80% of all renewable antibodies, but some top-cited polyclonal antibodies may not be available through these partners.

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 ADNP only a brief overview of the protein’s function and its relevance in disease is provided. ADNP 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. 14 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 Protocol Exchange, a preprint server (DOI: 10.21203/rs.3.pex-2607/v1). 14 Brief descriptions of the experimental setup used to carry out this study can be found below.

Cell lines and antibodies

Cell lines used and primary antibodies tested in this study are listed in Tables 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. 19, 20 DMS 53 cells were treated with the ON-TARGETplus Human ADNP siRNA from Horizon Discovery, cat. number L-012857-01-0005. Ctrl DMS 53 cells were treated with the ON-TARGETplus Non-targeting Control Pool, cat. number D-001810-10-05. Lipofectamine RNAiMAX (Thermo Fisher Scientific, cat. number 13778030) was used to transfect the siRNA following the manufacturer’s protocol.

Table 1. Summary of the cell lines used.

Institution Catalog number RRID (Cellosaurus) Cell line Genotype
ATCC CRL-2062 CVCL_1177 DMS 53 WT
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Table 2. Summary of the ADNP antibodies tested.

Company Catalog number Lot number RRID (Antibody Registry) Clonality Clone ID Host Concentration (μg/μL) Vendors recommended applications
Abcam ab300114 ** 1074869-4 AB_3097704 recombinant mono EPR25434-4 rabbit 0.54 Wb, IP, IF
ABclonal A4546 0092990201 AB_2765746 polyclonal - rabbit 0.70 Wb, IF
Bio-Techne (Novus Biologicals) NBP1-89236 000036064 AB_11008573 polyclonal - rabbit 0.10 Wb, IF
Bio-Techne (Novus Biologicals) NBP2-97747 HD12JL1810 AB_3094832 polyclonal - rabbit n/a Wb, IP, IF
Proteintech 17987-1-AP 00040236 AB_2222383 polyclonal - rabbit 0.60 Wb, IF
Thermo Fisher Scientific 702911 ** 2485913 AB_2848220 recombinant mono 16H17L68 rabbit 0.50 Wb, IF
Thermo Fisher Scientific PA5-52286 YE3914208 AB_2637725 polyclonal - rabbit 0.10 Wb, IF
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Wb = western blot; IP = immunoprecipitation; IF = immunofluorescence.

**

Recombinant antibody, n/a=not available.

Peroxidase-conjugated goat anti-rabbit antibody is from Thermo Fisher Scientific, cat. number 65-6120. Alexa-555-conjugated goat anti-rabbit secondary antibody is from Thermo Fisher Scientific, cat. number A-21429. Peroxidase-conjugated Protein A for IP detection is from Cell Signaling Technology, cat. number 12291.

Antibody screening by western blot

DMS 53 ctrl and ADNP KD cells were collected in RIPA buffer (25mM 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 1× protease inhibitor cocktail mix (MilliporeSigma, cat. number P8340). Lysates were sonicated briefly and incubated 30 min on ice. Lysates were spun at ~110,000 × 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 at a dilution of ~0.2 μg/ml 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) or Clarity Western ECL Substrate (Bio-Rad, cat. number 1705061) 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 2.0 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- (rabbit antibodies) (Thermo Fisher Scientific, cat. number 10002D). Antibody NBP2-97747 is at an unknown concentration, and 20 μl were tested in the IP. Tubes were rocked for ~1 h at 4°C followed by two washes to remove unbound antibodies.

DMS 53 WT 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 × g for 15 min at 4°C. 0.5 ml aliquots at 2 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. Protein A:HRP was used as a secondary detection system at a concentration of 0.5 μg/ml.

Antibody screening by immunofluorescence

DMS 53 ctrl and ADNP KD cells were labelled with a green and a far-red fluorescence dye, respectively (Thermo Fisher Scientific, cat. number C2925 and C34565). The nuclei were labelled with DAPI (Thermo Fisher Scientific, cat. Number D3571) fluorescent stain. Ctrl and KD cells were plated on 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 oC, 5% CO 2. Cells were fixed in 4% paraformaldehyde (PFA) (VWR, cat. number 100503-917) in phosphate buffered saline (PBS) (Wisent, cat. number 311-010-CL). Cells were permeabilized in PBS with 0,1% Triton X-100 (Thermo Fisher Scientific, cat. number BP151-500) for 10 min at room temperature and blocked with PBS with 5% BSA, 5% goat serum (Gibco, cat. number 16210-064) and 0.01% Triton X-100 for 30 min at room temperature. Cells were incubated with IF buffer (PBS, 5% BSA, 0,01% Triton X-100) containing the primary ADNP antibodies overnight at 4°C. Cells were then washed 3 × 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 μg/ml for 1 hr at room temperature with DAPI. Cells were washed 3 × 10 min with IF buffer and once with PBS.

Images were acquired on an ImageXpress micro confocal high-content microscopy system (Molecular Devices), using a 20× NA 0.95 water immersion 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 × 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. 21 Figures were assembled with Adobe Illustrator.

Acknowledgment

We would like to thank the NeuroSGC/YCharOS/EDDU collaborative group for their important contribution to the creation of an open scientific ecosystem of antibody manufacturers and KO cell line suppliers, for the development of community-agreed protocols, and for their shared ideas, resources, and collaboration. Members of the group can be found below. We would also like to thank the Advanced BioImaging Facility (ABIF) consortium for their image analysis pipeline development and conduction (RRID:SCR_017697). Members of each group can be found below.

NeuroSGC/YCharOS/EDDU collaborative group: Thomas M. Durcan, Aled M. Edwards, Peter S. McPherson, Chetan Raina and Wolfgang Reintsch

ABIF consortium: Claire M. Brown and Joel Ryan

Thank you to the Structural Genomics Consortium, a registered charity (no. 1097737), for your support on this project. The Structural Genomics Consortium receives funding from Bayer AG, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute (grant no. OGI-196), the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no. 875510), Janssen, Merck KGaA (also known as EMD in Canada and the United States), Pfizer and Takeda.

Funding Statement

This work was supported by a grant from the Simons Foundation Autism Research Initiative (SFARI). The Government of Canada through Genome Canada, Genome Quebec, and Ontario Genomics (grant no. OGI-210). RA is supported by a Mitacs fellowship.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

[version 1; peer review: 2 approved

Data availability

Underlying data

Zenodo: Antibody Characterization Report for ADNP, https://doi.org/10.5281/zenodo.14366335. 22

Zenodo: Dataset for the ADNP antibody screening study, https://doi.org/10.5281/zenodo.14423002. 23

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

References

  • 1. Mandel S, Gozes I: Activity-dependent neuroprotective protein constitutes a novel element in the SWI/SNF chromatin remodeling complex. J. Biol. Chem. 2007;282(47):34448–34456. 10.1074/jbc.M704756200 [DOI] [PubMed] [Google Scholar]
  • 2. Zamostiano R, Pinhasov A, Gelber E, et al. : Cloning and characterization of the human activity-dependent neuroprotective protein. J. Biol. Chem. 2001;276(1):708–714. 10.1074/jbc.M007416200 [DOI] [PubMed] [Google Scholar]
  • 3. Jo YS, Kim MS, Yoo NJ, et al. : ADNP encoding a transcription factor interacting with BAF complexes exhibits frameshift mutations in gastric and colorectal cancers. Scand. J. Gastroenterol. 2016;51(10):1269–1271. 10.1080/00365521.2016.1193220 [DOI] [PubMed] [Google Scholar]
  • 4. Bassan M, Zamostiano R, Davidson A, et al. : Complete sequence of a novel protein containing a femtomolar-activity-dependent neuroprotective peptide. J. Neurochem. 1999;72(3):1283–1293. 10.1046/j.1471-4159.1999.0721283.x [DOI] [PubMed] [Google Scholar]
  • 5. Pinhasov A, Mandel S, Torchinsky A, et al. : Activity-dependent neuroprotective protein: a novel gene essential for brain formation. Brain Res. Dev. Brain Res. 2003;144(1):83–90. 10.1016/S0165-3806(03)00162-7 [DOI] [PubMed] [Google Scholar]
  • 6. Gozes I: ADNP Regulates Cognition: A Multitasking Protein. Front. Neurosci. 2018;12:873. 10.3389/fnins.2018.00873 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Vulih-Shultzman I, Pinhasov A, Mandel S, et al. : Activity-dependent neuroprotective protein snippet NAP reduces tau hyperphosphorylation and enhances learning in a novel transgenic mouse model. J. Pharmacol. Exp. Ther. 2007;323(2):438–449. 10.1124/jpet.107.129551 [DOI] [PubMed] [Google Scholar]
  • 8. D’Incal CP, Annear DJ, Elinck E, et al. : Loss-of-function of activity-dependent neuroprotective protein (ADNP) by a splice-acceptor site mutation causes Helsmoortel-Van der Aa syndrome. Eur. J. Hum. Genet. 2024;32(6):630–638. 10.1038/s41431-024-01556-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Helsmoortel C, Vulto-van Silfhout AT, Coe BP, et al. : A SWI/SNF-related autism syndrome caused by de novo mutations in ADNP. Nat. Genet. 2014;46(4):380–384. 10.1038/ng.2899 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Van Dijck A, Vandeweyer G, Kooy F: ADNP-Related Disorder. Adam MP, Feldman J, Mirzaa GM, et al., editors. GeneReviews((R)). Seattle (WA);1993. [PubMed] [Google Scholar]
  • 11. Ayoubi R, Ryan J, Biddle MS, et al. : Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications. elife. 2023;12:12. 10.7554/eLife.91645 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Carter AJ, Kraemer O, Zwick M, et al. : Target 2035: probing the human proteome. Drug Discov. Today. 2019;24(11):2111–2115. 10.1016/j.drudis.2019.06.020 [DOI] [PubMed] [Google Scholar]
  • 13. Licciardello MP, Workman P: The era of high-quality chemical probes. RSC Med Chem. 2022;13(12):1446–1459. 10.1039/D2MD00291D [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Ayoubi R, Ryan J, Bolivar SG, et al. : A consensus platform for antibody characterization (Version 1). Protocol Exchange. 2024. [DOI] [PubMed] [Google Scholar]
  • 15. Biddle MS, Virk HS: YCharOS open antibody characterisation data: Lessons learned and progress made. F1000Res. 2023;12(12):1344. 10.12688/f1000research.141719.1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Laflamme C, McKeever PM, Kumar R, et al. : Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. elife. 2019;8:8. 10.7554/eLife.48363 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Fire A, Xu S, Montgomery MK, et al. : Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391(6669):806–811. 10.1038/35888 [DOI] [PubMed] [Google Scholar]
  • 18. Dana H, Chalbatani GM, Mahmoodzadeh H, et al. : Molecular Mechanisms and Biological Functions of siRNA. Int. J. Biomed. Sci. 2017;13(2):48–57. 10.59566/IJBS.2017.13048 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Bandrowski A, Pairish M, Eckmann P, et al. : The Antibody Registry: ten years of registering antibodies. Nucleic Acids Res. 2023;51(D1):D358–D367. 10.1093/nar/gkac927 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Bairoch A: The Cellosaurus, a Cell-Line Knowledge Resource. J. Biomol. Tech. 2018;29(2):25–38. 10.7171/jbt.18-2902-002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Stringer C, Wang T, Michaelos M, et al. : Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods. 2021;18(1):100–106. 10.1038/s41592-020-01018-x [DOI] [PubMed] [Google Scholar]
  • 22. Ruiz Moleon V, Alende C, Fotouhi M, et al. : A guide to selecting high-performing antibodies for ADNP (UniProt ID: Q9H2P0) for use in western blot, immunoprecipitation, and immunofluorescence. 2024. 10.5281/zenodo.14366335 [DOI] [PMC free article] [PubMed]
  • 23. Laflamme C: Dataset for the ADNP antibody screening study.[Data set]. Zenodo. 2024. 10.5281/zenodo.14423002 [DOI]
F1000Res. 2025 Feb 12. doi: 10.5256/f1000research.175953.r352668

Reviewer response for version 1

Illana Gozes 1, Maram Ganaiem 2, Artur Galushkin 2

Review of “A Guide to Selecting High-Performing Antibodies for ADNP”

Overview

The article under review provides a characterization of seven commercially available antibodies targeting ADNP. The study aims to address the widespread issue of antibody reproducibility by evaluating antibody performance on western blots, immunoprecipitations, and immunofluorescence. This open-access initiative seeks to guide researchers in selecting reliable antibodies for their specific experimental applications.

Previously, we produced our own antibodies as well as used different commercial ADNP antibodies including for example the F-9 mouse monoclonal IgG1s (sc-376674, Santa Cruz) recognizing amino acids 1-138, mapped to the N-terminus of human ADNP (please see selected citations). These antibodies were not used for comparison here.

The antibodies used in the current paper bring forth two recombinant monoclonal ADNP antibodies (rabbit), which seem to be of further interest in terms of specificity in western blots. Antibody 702911** (commercially available here - Ref:6 - was used at 1/500 for further validation of the immunoprecipitation results. The picture for the other recombinant antibody used (ab300114) looks very similar to the picture depicted on the commercial site: https://www.abcam.com/en-us/products/primary-antibodies/adnp-antibody-epr25434-4-ab300114?srsltid=AfmBOorKkVs4moqf5KOeCqUNZLbuTpM7BipS36RTsmMo9bpSixSbPt8k

Interestingly, the ADNP molecular weight using the immunoprecipitation buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) seems lower than that seen for in the cell lysates utilizing the RIPA buffer (25mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS), suggesting instability of the ADNP protein, although in both cases supplementation with protease inhibitors was mentioned – i.e. 1x protease inhibitor cocktail mix (MilliporeSigma, cat. number P8340). In this respect, the stability of the ADNP protein was recently addressed (please see selected citations).

Key Findings and Strengths

The authors systematically evaluated ADNP antibodies using a standardized protocol in a DMS 53 cell line (DMS 53 is a  cell line exhibiting epithelial morphology that was isolated in 1974 from the lung of a 54-year-old, White male patient with carcinoma. This cell line was deposited by OS Pettengill, G Sorenson, and can be used in cancer research). To address specificity ADNP expression was knocked down using siRNA. As mentioned above, performance was assessed across three methodologies:

  • Western blot: Antibodies were screened for specificity based on expected molecular weights and signal intensity. Interestingly, the authors consistently detected a 135kD ADNP-immunoreactive band

  • Immunoprecipitation: The ability of antibodies to pull down ADNP from cell lysates was examined.

  • Immunofluorescence: Quantitative imaging revealed differential staining in control versus knockdown cells.

The study's strengths lie in its rigorous methodology, adherence to reproducibility standards, and commitment to Open Science. The authors provide openly available protocols and raw data, encouraging independent validation and application in diverse experimental contexts.

Limitations and Gaps

The authors do not perform replicates, citing the use of multiple antibodies targeting various epitopes as the reason for this choice. They explain that validation of a single antibody provides sufficient confirmation of target specificity in the selected cell line and knockdown conditions. While this approach streamlines the process, it could reduce confidence in reproducibility, particularly for users requiring robust replication. The molecular weight obtained depends on post-translational modifications as well as protein sources, which could be further discussed.

Methodological Assessment

While the use of a single cell line (DMS 53) ensures a controlled comparison, it introduces potential limitations in generalizability.

Antibody Validation

The characterization revealed both high- and low-performing antibodies, allowing researchers to make informed choices. However, the article refrains from recommending specific antibodies, placing the onus on users to interpret the data. This non-prescriptive approach aligns with Open Science principles but might challenge researchers unfamiliar with antibody validation intricacies.

Suggestions for Improvement

To increase the impact of future studies, the authors could:

  • Include additional cell lines and tissue samples to validate findings across biological contexts (e.g., brain tissue).

  • Conduct replicative studies to reinforce confidence in antibody reproducibility.

  • Provide more detailed guidance on interpreting banding patterns and staining artifacts in western blot and immunofluorescence.

Conclusion

This study offers a valuable resource for researchers investigating ADNP and related pathways. Despite some methodological constraints, it sets a high standard for antibody characterization. Incorporating these findings into experiments could streamline the selection of reliable tools and enhance the reproducibility of research.

The recombinant antibodies show the highest specificity to ADNP and look promising to use.

In writing this review I (Professor Illana Gozes) consulted with my two excellent PhD students as follows. Artur Galushkin is supported by a Marie Skłodowska-Curie TClock4AD fellowship aimed to discover pharmacological drugs that improve Alzheimer’s disease disrupted circadian rhythmicity & student training (double doctorate degrees). Project#: 101072895, EUROPEAN RESEARCH EXECUTIVE AGENCY (REA) REA) and Maram Ganaiem is supported by a Neubauer Family Foundation Student Scholarship at Tel Aviv University.

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Molecular and translational neuroscience/neurochemistry. Expert (discovorer) of ADNP.

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

References

  • 1. : Distinct Impairments Characterizing Different ADNP Mutants Reveal Aberrant Cytoplasmic-Nuclear Crosstalk. Cells .2022;11(19) : 10.3390/cells11192994 10.3390/cells11192994 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. : Introducing ADNP and SIRT1 as new partners regulating microtubules and histone methylation. Mol Psychiatry .2021;26(11) : 10.1038/s41380-021-01143-9 6550-6561 10.1038/s41380-021-01143-9 [DOI] [PubMed] [Google Scholar]
  • 3. : SH3- and actin-binding domains connect ADNP and SHANK3, revealing a fundamental shared mechanism underlying autism. Mol Psychiatry .2022;27(8) : 10.1038/s41380-022-01603-w 3316-3327 10.1038/s41380-022-01603-w [DOI] [PubMed] [Google Scholar]
  • 4. : Activity-dependent neuroprotective protein (ADNP) differentially interacts with chromatin to regulate genes essential for embryogenesis. Dev Biol .2007;303(2) : 10.1016/j.ydbio.2006.11.039 814-24 10.1016/j.ydbio.2006.11.039 [DOI] [PubMed] [Google Scholar]
  • 5. : Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients. Sci Rep .2024;14(1) : 10.1038/s41598-024-65608-x 14710 10.1038/s41598-024-65608-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. : Toxicity of pyrolysis products of thermal-resistant plastics including polyamide and polyester. Nihon Eiseigaku Zasshi .1978;33(2) : 10.1265/jjh.33.450 450-8 10.1265/jjh.33.450 [DOI] [PubMed] [Google Scholar]
F1000Res. 2025 Jan 15. doi: 10.5256/f1000research.175953.r352667

Reviewer response for version 1

Christian Peter Moritz 1

This report evaluates a study focused on characterizing commercial ADNP antibodies across various applications, contributing to a broader initiative aimed at addressing antibody reproducibility issues.

The project is highly relevant, addressing a critical issue in biomedical research by tackling the reproducibility crisis in antibody-based experiments. The use of standardized protocols in knockout and isogenic parental controls highlights a rigorous and transparent approach, offering valuable insights and an open-access resource for the scientific community.

I see options for the following improvements:

Major Points:

  1. Figure 2: If I am not mistaken, the band pattern in all of the panels’ SM (start material) lanes should match the pattern observed in the control lane for antibody 702911 in Figure 1—specifically, a strong band around 135 kDa and a very weak band slightly below it. However, the pattern and the molecular weight of the main band differ, suggesting significant technical variations among the experiments. Can we rule out technical variability among the tested antibodies? Are the observed differences truly attributable to the antibodies themselves?

  2. Precipitation Studies: For the precipitation studies, the authors used antibody 702911 to detect the precipitated proteins (“a specific ADNP antibody identified previously (refer to Figure 1) was selected”).

    However, in my view, antibody ab300114 appears to be more specific in Figure 1. I would like to understand why the authors chose antibody 702911 over ab300114

  3. Competing Interests: Regarding their general YCharOS project, the authors might critically consider whether there are potential flaws in the study design due to the fact that the industry partner provided both the antibodies and the corresponding KO cell line. This creates a situation where the commercial antibody is being tested using a tool supplied by the same company. Are there any potential issues with this approach that should be discussed?

    I am aware that this criticism is rather for the general project as specifically for this study as the authors prepared their own Knock-down instead of using a company's Knock-out line if I understand it correctly. 

Minor Points:

  1. Title and Abstract: I suggest introducing abbreviations—even for protein names like ADNP—the first time they are mentioned.

  2. While many reviewers recommend using housekeeping proteins as loading controls for Western blots, I appreciate that the authors have opted for a total protein staining technique (Ponceau Red) to provide a more transparent representation of the loaded protein amounts (cf. 10.1002/pmic.201600189). This is just a positive note; no changes are necessary.

My best wishes to the authors for their valuable project.

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Partly

Reviewer Expertise:

Antibodies, western blot, immunoprecipitation, immunofluorescence, ELISA, Proteomics, Neuropathies

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.

F1000Res. 2025 Jan 10. doi: 10.5256/f1000research.175953.r352666

Reviewer response for version 1

Richard O'Kennedy 1

1. This paper presents data on the binding characteristics of a set of commercially available antibodies that bind to the protein ADNP (UniProt ID:Q9H2PO), as determined using a variety of techniques including western blotting, immunoprecipitation, and immunofluorescence employing standardized experimental devised protocols. Determinations were made  by comparing read-outs in knockout cell lines and isogenic parental controls.

2. The paper is designed to present the resultant data to enable researchers to compare the  characteristics of the antibodies and their performance, in order to help researchers choose the most ideal/appropriate antibody for their particular applications.

3. For the most part the data is clearly set out but it would have helped if the legends to the figures were more explanatory and included the full-names associated with abbreviations.

4. The authors cite the approaches used in the methodologies employed. In addition, a section of the paper critically analyses the approaches and sets out possible shortcomings that the approaches may inherently have. This is very useful and would be particularly valuable if more papers had such evaluations generally.

5. Ensuring that the binding characteristics/specificity of antibodies is as well defined as possible is very important and this paper certainly assists in this in relation to the target protein. Fundamental questions have been raised about data generated where the antibodies were poorly characterized, and had specificity issues that were later realized, which may have been avoided by more intense and careful binding characterization studies.

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Antibody production, characterisation and use with particular emphasis on diagnostics.

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.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Citations

    1. Laflamme C: Dataset for the ADNP antibody screening study.[Data set]. Zenodo. 2024. 10.5281/zenodo.14423002 [DOI]

    Data Availability Statement

    Underlying data

    Zenodo: Antibody Characterization Report for ADNP, https://doi.org/10.5281/zenodo.14366335. 22

    Zenodo: Dataset for the ADNP antibody screening study, https://doi.org/10.5281/zenodo.14423002. 23

    Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

    Articles from F1000Research are provided here courtesy of F1000 Research Ltd

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