A guide to selecting high-performing antibodies for Secreted frizzled-related protein 1 (sFRP-1) for use in Western Blot and immunoprecipitation

Riham Ayoubi; Kathleen Southern; Carl Laflamme; NeuroSGC/YCharOS collaborative group

doi:10.12688/f1000research.130991.2

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Revised

A guide to selecting high-performing antibodies for Secreted frizzled-related protein 1 (sFRP-1) for use in Western Blot and immunoprecipitation

[version 2; peer review: 1 approved, 2 approved with reservations]

Previous Title 'The identification of high-performing antibodies for Secreted frizzled-related protein 1 (sFRP-1) for use in Western Blot and immunoprecipitation'

Riham Ayoubi¹, Kathleen Southern¹, Carl Laflamme ¹, NeuroSGC/YCharOS collaborative group

PUBLISHED 02 Apr 2024

Author details Author details

¹ Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Quebec, H3A 2B4, Canada

Riham Ayoubi
Roles: Investigation, Visualization, Writing – Review & Editing

Kathleen Southern
Roles: Writing – Original Draft Preparation, Writing – Review & Editing

Carl Laflamme
Roles: Conceptualization, Data Curation, Funding Acquisition, Methodology, Project Administration, Resources, Supervision, Validation, Visualization, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the YCharOS (Antibody Characterization through Open Science) gateway.

This article is included in the Cell & Molecular Biology gateway.

Abstract

Secreted frizzled-related protein 1 (sFRP-1) is a secreted protein, belonging to the secreted glycoprotein SFRP family. As a modulator of the Wnt/β-catenin signalling pathway, sFRP-1 has implications in human cancers and neurological diseases. If the community had access to well-characterized anti-sFRP-1 antibodies, the reproducibility of sFRP-1 research would be enhanced. In this study, we characterized 11 sFRP-1 commercial antibodies for Western Blot and immunoprecipitation, 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 the antibody reproducibility issue 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

Uniprot ID Q8N474, SFRP1, Secreted frizzled-related protein 1, antibody characterization, antibody validation, Western Blot, immunoprecipitation

Corresponding author: Carl Laflamme

Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam- Abclonal -Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -Genetex – Horizon Discovery – Proteintech – Synaptic Systems -Thermo Fisher Scientific.

Grant information: This work was supported by a grant from the Emory-Sage-SGC TREAT-AD center established by the National Institutes of Aging (no. U54AG065187). The Government of Canada through Genome Canada and Ontario Genomics (no. OGI-210). The Structural Genomics Consortium is a registered charity (no. 1097737) that receives funds from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute (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. RA is supported by a Mitacs postdoctoral fellowship.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Ayoubi R 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: Ayoubi R, Southern K, Laflamme C and NeuroSGC/YCharOS collaborative group. A guide to selecting high-performing antibodies for Secreted frizzled-related protein 1 (sFRP-1) for use in Western Blot and immunoprecipitation [version 2; peer review: 1 approved, 2 approved with reservations]. F1000Research 2024, 12:291 (https://doi.org/10.12688/f1000research.130991.2) First published: 16 Mar 2023, 12:291 (https://doi.org/10.12688/f1000research.130991.1) Latest published: 02 Apr 2024, 12:291 (https://doi.org/10.12688/f1000research.130991.2)

Revised Amendments from Version 1

In this new version of the article, statements have been added to the Abstract, Introduction and Results & Discussion sections to ensure the YCharOS initiative and goals are clearly defined to prevent misinterpretation.

See the authors' detailed response to the review by Viyatprajna Acharya
See the authors' detailed response to the review by Oi Wah Liew

Introduction

SFRP-1 is a secreted protein belonging to the secreted frizzled-related protein (SFRP) family.¹ Having an amino acid terminal cysteine-rich domain homologous to the putative Wnt-binding domain of frizzled receptors, sFRP-1 functions as a modulator of the Wnt/β-catenin signalling pathway that is essential to multiple cellular processes.²^,³

Dysregulated Wnt/β-catenin activity levels play a role in various disease processes.⁴ Epigenetic silencing of SFRP1 elevates Wnt/β-catenin activity, which has been linked to cancer.²^,⁴ Conversely, high concentrations of sFRP-1 suppresses Wnt/β-catenin activity, which is predicted to have implications in neurological diseases, such as Alzheimer’s disease (AD).¹^,³^–⁵ Targeting sFRP-1 to restore the Wnt/β-catenin signalling pathway is of current interest in AD research to discover novel therapies.³ Mechanistic studies would be greatly facilitated with the availability of high-quality antibodies.

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.⁶^–⁸ Here, we compared the performance of a range of commercially available antibodies for sFRP-1 use in Western Blot and immunoprecipitation. This article serves as a valuable guide to help researchers select high-quality antibodies for their specific needs, facilitating the biochemical and cellular assessment of sFRP-1 properties and function.

Results and discussion

Our standard protocol involves comparing readouts from wild-type and knockout (KO) cells.⁹^,¹⁰ 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 log₂ (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 sFRP-1 transcript at RNA levels above the average range of cancer cells analyzed. Parental and SFRP1 knockout A549 cells were obtained from Abcam (Table 1).

Table 1. Summary of the cell lines used.

Institution	Catalog number	RRID (Cellosaurus)	Cell line	Genotype
Abcam	ab275463	CVCL_0023	A549	WT
Abcam	ab277906	CVCL_B2Q1	A549	SFRP1 KO

SFRP-1 is predicted to be a secreted protein. Accordingly, we collected concentrated culture media from both wild-type and SFRP1 KO cells and used the conditioned media to probe the performance of the antibodies (Table 2) side-by-side by Western blot and immunoprecipitation. The profiles of each of the antibodies are shown in Figures 1 and 2. The datasets can be found as Underlying data.¹³^,¹⁴

Table 2. Summary of the Secreted frizzled-related protein 1 antibodies tested.

Company	Catalog number	Lot number	RRID (Antibody Registry)	Clonality	Clone ID	Host	Concentration (μg/μl)	Vendors recommended applications
Thermo Fisher Scientific	MA5-32675**	WJ3417745	AB_2809952	recombinant-mono	JA11-68	rabbit	1.00	Wb
Thermo Fisher Scientific	MA5-38193**	WJ3417799B	AB_2898110	recombinant-mono	ARC1683	rabbit	0.88	Wb
GeneTex	GTX24193	822102161	AB_370619	polyclonal	-	rabbit	1.00	Wb, IF
GeneTex	GTX102371	39911	AB_1951886	polyclonal	-	rabbit	0.33	Wb
ABclonal	A9656**	4000001683	AB_2863750	recombinant-mono	ARC1683	rabbit	0.88	Wb
ABclonal	A2911	31570101	AB_2764730	polyclonal	-	rabbit	2.68	Wb
Bio-Techne	AF1384	IRQ1020021	AB_2285831	polyclonal	-	goat	0.20	Wb, IF
Abcam	ab4193	GR3345186-4	AB_304357	polyclonal	-	rabbit	1.00	Wb, IF
Abcam	ab126613**	GR3350102-3	AB_11128257	recombinant-mono	EPR7003	rabbit	0.41	Wb
Abcam	ab267466**	GR3321068-3	AB_2904616	recombinant-mono	EPR23092-253	rabbit	0.46	Wb, IP
Abcam	ab84003	GR42188-1	AB_10670402	polyclonal	-	rabbit	1.00	Wb

** Recombinant antibody.

Figure 1. Secreted frizzled-related protein 1 antibody screening by Western Blot on culture media.

A549 (WT and SFRP1 KO) were treated with Brefeldin A at 3.0 μg/ml for 18 hrs. 50 μg of protein from concentrated culture media were processed for Western Blot with the indicated sFRP-1 antibodies. The Ponceau stained transfers of each blot are shown. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Antibody dilution used: MA5-32675** at 1/500; MA5-38193** at 1/500; GTX24193 at 1/1000; GTX102371 at 1/1000; A9656** at 1/1000; A2911 at 1/1000; AF1384 at 1/500; ab4193 at 1/500; ab126613** at 1/1000; ab267466** at 1/1000; ab84003 at 1/1000. Predicted band size: 35 kDa. **Recombinant antibody.

Figure 2. Secreted frizzled-related protein 1 antibody screening by immunoprecipitation on culture media.

Immunoprecipitation was performed on concentrate culture media using 1.0 μg of the indicated sFRP-1 antibodies pre-coupled to either protein A or protein G magnetic beads. Samples were washed and processed for Western Blot with the indicated sFRP-1 antibody. For Western Blot, MA5-38193** was used at 1/500, A9656** at 1/1000 and ab126613** at 1/1000. The Ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM = 10% starting material; UB = 10% unbound fraction; IP = immunoprecipitated; LC = light chain. **Recombinant antibody.

In conclusion, we have screened eleven sFRP-1 commercial antibodies by Western Blot and immunoprecipitation. Several high-performing antibodies that successfully detect sFRP-1 under our standardized experimental conditions. 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. However, 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.

Methods

Antibodies

All sFRP-1 antibodies are listed in Table 2. Peroxidase-conjugated goat anti-rabbit and donkey anti-goat antibodies are from Thermo Fisher Scientific (cat. number 65-6120 and A15999).

Cell culture

A549 WT and SFRP1 KO used are listed in Table 1. Cells were cultured in DMEM high-glucose (GE Healthcare cat. number SH30081.01) containing 10% fetal bovine serum (Wisent, cat. number 080450), 2 mM L-glutamate (Wisent cat. number 609065), 100 IU penicillin and 100 μg/ml streptomycin (Wisent cat. number 450201). Cells were starved in DMEM high-glucose containing L-glutamate and penicillin/streptomycin.

Antibody screening by Western Blot on culture media

A549 cells (WT and SFRP1 KO) were washed 3× with PBS and starved for ~18 hrs. Culture media were collected and centrifuged for 10 min at 500 × g to eliminate cells and larger contaminants, then for 10 min at 4500 × g to eliminate smaller contaminants. Culture media were concentrated by centrifuging at 4000 × g for 10 min using Amicon Ultra-15 Centrifugal Filter Units with a membrane NMWL of 10 kDa (MilliporeSigma cat. number UFC901024).

Western Blots were performed as described in our standard operating procedure.¹¹ Western Blots were performed with large 8-16% gradient polyacrylamide gels and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau staining which is scanned to show together with individual Western blot. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated overnight at 4°C with 5% bovine serum albumin in TBS with 0,1% Tween 20 (TBST). 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 more washes with TBST. Membranes were incubated with ECL from Pierce (cat. number 32106) prior to detection with HyBlot CL autoradiography films from Denville (cat. number 1159T41).

Antibody screening by immunoprecipitation on culture media

Immunoprecipitation was performed as described in our standard operating procedure.¹² Antibody-bead conjugates were prepared by adding 1 μg of antibody to 500 ul of Pierce IP 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 goat antibodies) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively). Pierce IP Lysis Buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) was supplemented with the Halt Protease Inhibitor Cocktail 100X from Thermo Fisher Scientific (cat. number 78446) at a final concentration of 1×. Tubes were rocked for ~2 hrs at 4°C followed by two washes to remove unbound antibodies.

Starved A549 WT culture media were concentrated as described above. 1 ml aliquots at 0.5 mg/ml of lysate were incubated with an antibody-bead conjugate for ~2 hrs at 4°C. Following centrifugation, the unbound fractions were collected, and beads were subsequently washed three times with 1.0 ml of IP Lysis Buffer and processed for SDS-PAGE and Western Blot on 8-16% polyacrylamide gels. Prot-A: HRP (MilliporeSigma, cat. number P8651) was used as a secondary detection system at a dilution of 0.4 μg/ml.

Data availability

Underlying data

Zenodo: Antibody Characterization Report for Secreted frizzled-related protein 1, https://doi.org/10.5281/zenodo.6370454.¹³

Zenodo: Dataset for the Secreted frizzled-related protein 1 antibody screening study, https://doi.org/10.5281/zenodo.7571170.¹⁴

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

Acknowledgments

We’d like to thank the NeuroSGC/YCharOS collaborative group for their important contribution to the creation of an open scientific ecosystem of antibody manufacturers and knockout cell line suppliers as well as the development of community-agreed protocols. Members of the group can be found below.

NeuroSGC/YCharOS collaborative group: Riham Ayoubi, Aled M. Edwards, Carl Laflamme, Peter S. McPherson, Chetan Raina and Kathleen Southern.

An earlier version of this article can be found on Zenodo (doi: 10.5281/zenodo.6370454).

References

1. Liang CJ, Wang ZW, Chang YW, et al.: SFRPs Are Biphasic Modulators of Wnt-Signaling-Elicited Cancer Stem Cell Properties beyond Extracellular Control. Cell Rep. 2019; 28(6): 1511–25.e5. PubMed Abstract | Publisher Full Text
2. Baharudin R, Tieng FYF, Lee LH, et al.: Epigenetics of SFRP1: The Dual Roles in Human Cancers. Cancers (Basel). 2020; 12(2). PubMed Abstract | Publisher Full Text | Free Full Text
3. Jia L, Piña-Crespo J, Li Y: Restoring Wnt/β-catenin signaling is a promising therapeutic strategy for Alzheimer's disease. Mol. Brain. 2019; 12(1): 104. PubMed Abstract | Publisher Full Text | Free Full Text
4. Zimmerman ZF, Moon RT, Chien AJ: Targeting Wnt pathways in disease. Cold Spring Harb. Perspect. Biol. 2012; 4(11). PubMed Abstract | Publisher Full Text | Free Full Text
5. Johnson ECB, Carter EK, Dammer EB, et al.: Large-scale deep multi-layer analysis of Alzheimer's disease brain reveals strong proteomic disease-related changes not observed at the RNA level. Nat. Neurosci. 2022; 25(2): 213–225. PubMed Abstract | Publisher Full Text | Free Full Text
6. Ayoubi R, et al.: Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications. Elife. 2023; 12: RP91645. PubMed Abstract | Publisher Full Text | Free Full Text
7. Licciardello MP, Workman P: The era of high-quality chemical probes. RSC Med. Chem. 2022; 13(12): 1446–1459. PubMed Abstract | Publisher Full Text | Free Full Text
8. Carter AJ, et al.: Target 2035: probing the human proteome. Drug Discov. Today. 2019; 24(11): 2111–2115. PubMed Abstract | Publisher Full Text
9. 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. Publisher Full Text
10. Alshafie W, Fotouhi M, Shlaifer I, et al.: Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence. F1000Res. 2022; 11: 977. Publisher Full Text
11. Ayoubi R, McPherson PS, Laflamme C: Antibody Screening by Immunoblot.2021. Publisher Full Text
12. Ayoubi R, Fotouhi M, McPherson P, et al.: Antibody screening by Immunoprecitation.2021. Publisher Full Text
13. Ayoubi R, McPherson, Peter S, et al.: Antibody Characterization Report for Secreted frizzled-related protein 1. [Dataset]. 2022. Publisher Full Text
14. Laflamme C: Dataset for the Secreted frizzled-related protein 1 antibody screening study. [Dataset]. Zenodo. 2023. Publisher Full Text

Comments on this article Comments (0)

Version 2

VERSION 2 PUBLISHED 16 Mar 2023

Author details Author details

¹ Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Quebec, H3A 2B4, Canada

Riham Ayoubi
Roles: Investigation, Visualization, Writing – Review & Editing

Kathleen Southern
Roles: Writing – Original Draft Preparation, Writing – Review & Editing

Carl Laflamme
Roles: Conceptualization, Data Curation, Funding Acquisition, Methodology, Project Administration, Resources, Supervision, Validation, Visualization, Writing – Review & Editing

Competing interests

For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam- Abclonal -Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -Genetex – Horizon Discovery – Proteintech – Synaptic Systems -Thermo Fisher Scientific.

Grant information

This work was supported by a grant from the Emory-Sage-SGC TREAT-AD center established by the National Institutes of Aging (no. U54AG065187). The Government of Canada through Genome Canada and Ontario Genomics (no. OGI-210). The Structural Genomics Consortium is a registered charity (no. 1097737) that receives funds from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute (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. RA is supported by a Mitacs postdoctoral fellowship.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (2)

version 2

Revised

Published: 02 Apr 2024, 12:291

https://doi.org/10.12688/f1000research.130991.2

version 1

Published: 16 Mar 2023, 12:291

https://doi.org/10.12688/f1000research.130991.1

© 2024 Ayoubi R 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.

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Ayoubi R, Southern K, Laflamme C and NeuroSGC/YCharOS collaborative group. A guide to selecting high-performing antibodies for Secreted frizzled-related protein 1 (sFRP-1) for use in Western Blot and immunoprecipitation [version 2; peer review: 1 approved, 2 approved with reservations]. F1000Research 2024, 12:291 (https://doi.org/10.12688/f1000research.130991.2)

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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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions

Version 2

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PUBLISHED 02 Apr 2024

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Reviewer Report 23 Sep 2024

Lynne Howells, University of Leicester, Leicester, UK

Approved

https://doi.org/10.5256/f1000research.164273.r325242

The article describes a protocol that will facilitate the use of better antibody tools in the detection of the sFRP-1 protein via western blot and IP techniques.

The article is succinct, well written and will provide an enabler for researchers looking to analyse this specific protein.

Even though the article is short, there are a number of areas where 'lab slang' has crept in, which should be rectified.:

When saying cells were starved – specify serum starved, or say that media was subsequently replaced with serum-free media.
In the Ab screening by IP section – it says 500 uL instead of micro (mu symbol)
Millilitres should be mL rather than ml

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

Yes
Are the protocols appropriate and is the work technically sound?

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

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

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Cancer biology, cell signalling (WB, IP, FACS, IHC, IF)

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.

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Reviewer Report 18 Apr 2024

Oi Wah Liew, Cardiovascular Research Institute, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, Singapore, Singapore

Approved with Reservations

https://doi.org/10.5256/f1000research.164273.r261793

The authors have not addressed any of my concerns. However, I note that they have made a qualifying statement in their report that "any conclusions drawn remain relevant within the confines of the experimental set up and cell line used in this study." I accept that those in the field will be able to interpret the available data in the light of their expertise.

As such, I understand that the authors need only to provide information on why and how the data were created but do not need to include analyses or conclusions. Indeed, the authors did not address any of my concerns, some of which
requires additional experiments or an explanation of seemingly contradictory data between Figure 1 and 2. Since the instructions to authors is that analyses or conclusions are not required, I am not sure if I am asking more than is required of the authors. It would be great if the authors could at least provide a point-by-point rebuttal of my concerns in the comments section.

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Biomarkers, antibody generation/characterization and validation, antigen design, recombinant protein expression, genetic engineering, assay development.

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.

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Author Response 28 Jun 2024

Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

28 Jun 2024

Author Response

Please see below our response to your concerns, point-by-point. We hope these responses address any misunderstandings or hesitations you previously had, providing a clearer understanding of the manuscripts purpose, the ... Continue reading Please see below our response to your concerns, point-by-point. We hope these responses address any misunderstandings or hesitations you previously had, providing a clearer understanding of the manuscripts purpose, the YCharOS initiative as a whole and limitations we face in terms of data interpretation and our protocol.

1.       The authors selected the A549 cell line based on available data on high expression at transcript levels. However, high transcript levels do not necessarily translate to high protein expression. The A549 and A549 sFRP1 KO cell lines as well as some of the antibodies tested were from the same vendor. To have added confidence that the selected antibodies are recognizing endogenous sFRP-1 expressed in A549 cells, the authors should include a recombinant source of sFRP-1 from a different vendor in their western blots. In others words, each antibody is tested against endogenous sFRP-1 expressed in A549 cells, A549 SFRP1 KO (negative control) and recombinant sFRP-1 (eg R&D Systems cat no. 1384-SF).
Agreed, mRNA levels do not always reflect protein expression, however our results show that A549 is an appropriate cell line for antibody testing as the protein was very easily detected in the culture medium by western blot.
The antibodies tested are from five different vendors as shown in Table 2: Thermo Fisher Scientific, GeneTex, ABclonal, Bio-Techne and Abcam.
The purpose of this manuscript is to detect the endogenous protein in a relevant sample (culture medium because the protein is secreted). Testing the antibody’s ability to recognize the recombinant proteins is screened internally and routinely by the manufacturer but do not always mean recognition in biologically relevant samples that researchers may be interested in investigating. This study offers a valuable antibody screening in the appropriate sample and in the presence of the most agreed upon type of negative control, a KO.

2.       Human sFRP-1 is a member of the SFRP family. Since many of the antibodies selected are polyclonal in nature, the authors should assess potential cross-reactivity against other closely related family members eg human sFRP-2, sFRP-4 and sFRP-5. Sequence alignment between sFRP-1 against these family members shows stretches of 100% identical amino acids up to 22 amino acids long, portending potential cross-reactivity of anti-sFRP-1 polyclonal antibodies against these entities. Cross-reactivity of the recombinant monoclonal antibodies should also be tested since there is no epitope information for these antibodies.
Cross-reactivity against other closely related family members can only be assessed using other KO controls corresponding to the mentioned genes of interest which we do not have access to. For now, this work is only informative about antibody specificity against sFRP1.

3.       The results for GTX4193, GTX102371, AF1384, AB4193 and AB84003 in Figure 1 and 2 appear contradictory. In Figure 1, these antibodies could not detect sFRP-1 present in concentrated culture media collected from starved A549 cells by western blotting. However, these antibodies were able to detect sFRP-1 at ~35 kDa position in the starting material samples in Figure 2 which are essentially equivalent to the WT samples in Figure 1.
In Figure 2, as indicated in the legend, all starting material and immunoprecipitates corresponding to each antibody tested are western blotted with one antibody, MA5-38193. Since MA5-38193 detects sFRP-1 by WB (Figure 1), signal is visible in all SM lanes.

The detailed antibody characterization platform we have created, along with academic and industry researchers, is available on Protocol Exchange. https://doi.org/10.21203/rs.3.pex-2607/v1
Please see below our response to your concerns, point-by-point. We hope these responses address any misunderstandings or hesitations you previously had, providing a clearer understanding of the manuscripts purpose, the YCharOS initiative as a whole and limitations we face in terms of data interpretation and our protocol.

1.       The authors selected the A549 cell line based on available data on high expression at transcript levels. However, high transcript levels do not necessarily translate to high protein expression. The A549 and A549 sFRP1 KO cell lines as well as some of the antibodies tested were from the same vendor. To have added confidence that the selected antibodies are recognizing endogenous sFRP-1 expressed in A549 cells, the authors should include a recombinant source of sFRP-1 from a different vendor in their western blots. In others words, each antibody is tested against endogenous sFRP-1 expressed in A549 cells, A549 SFRP1 KO (negative control) and recombinant sFRP-1 (eg R&D Systems cat no. 1384-SF).
Agreed, mRNA levels do not always reflect protein expression, however our results show that A549 is an appropriate cell line for antibody testing as the protein was very easily detected in the culture medium by western blot.
The antibodies tested are from five different vendors as shown in Table 2: Thermo Fisher Scientific, GeneTex, ABclonal, Bio-Techne and Abcam.
The purpose of this manuscript is to detect the endogenous protein in a relevant sample (culture medium because the protein is secreted). Testing the antibody’s ability to recognize the recombinant proteins is screened internally and routinely by the manufacturer but do not always mean recognition in biologically relevant samples that researchers may be interested in investigating. This study offers a valuable antibody screening in the appropriate sample and in the presence of the most agreed upon type of negative control, a KO.

2.       Human sFRP-1 is a member of the SFRP family. Since many of the antibodies selected are polyclonal in nature, the authors should assess potential cross-reactivity against other closely related family members eg human sFRP-2, sFRP-4 and sFRP-5. Sequence alignment between sFRP-1 against these family members shows stretches of 100% identical amino acids up to 22 amino acids long, portending potential cross-reactivity of anti-sFRP-1 polyclonal antibodies against these entities. Cross-reactivity of the recombinant monoclonal antibodies should also be tested since there is no epitope information for these antibodies.
Cross-reactivity against other closely related family members can only be assessed using other KO controls corresponding to the mentioned genes of interest which we do not have access to. For now, this work is only informative about antibody specificity against sFRP1.

3.       The results for GTX4193, GTX102371, AF1384, AB4193 and AB84003 in Figure 1 and 2 appear contradictory. In Figure 1, these antibodies could not detect sFRP-1 present in concentrated culture media collected from starved A549 cells by western blotting. However, these antibodies were able to detect sFRP-1 at ~35 kDa position in the starting material samples in Figure 2 which are essentially equivalent to the WT samples in Figure 1.
In Figure 2, as indicated in the legend, all starting material and immunoprecipitates corresponding to each antibody tested are western blotted with one antibody, MA5-38193. Since MA5-38193 detects sFRP-1 by WB (Figure 1), signal is visible in all SM lanes.

The detailed antibody characterization platform we have created, along with academic and industry researchers, is available on Protocol Exchange. https://doi.org/10.21203/rs.3.pex-2607/v1
Competing Interests: No competing interests were disclosed. Close
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COMMENTS ON THIS REPORT

Author Response 28 Jun 2024

Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

28 Jun 2024

Author Response

Please see below our response to your concerns, point-by-point. We hope these responses address any misunderstandings or hesitations you previously had, providing a clearer understanding of the manuscripts purpose, the ... Continue reading Please see below our response to your concerns, point-by-point. We hope these responses address any misunderstandings or hesitations you previously had, providing a clearer understanding of the manuscripts purpose, the YCharOS initiative as a whole and limitations we face in terms of data interpretation and our protocol.

1.       The authors selected the A549 cell line based on available data on high expression at transcript levels. However, high transcript levels do not necessarily translate to high protein expression. The A549 and A549 sFRP1 KO cell lines as well as some of the antibodies tested were from the same vendor. To have added confidence that the selected antibodies are recognizing endogenous sFRP-1 expressed in A549 cells, the authors should include a recombinant source of sFRP-1 from a different vendor in their western blots. In others words, each antibody is tested against endogenous sFRP-1 expressed in A549 cells, A549 SFRP1 KO (negative control) and recombinant sFRP-1 (eg R&D Systems cat no. 1384-SF).
Agreed, mRNA levels do not always reflect protein expression, however our results show that A549 is an appropriate cell line for antibody testing as the protein was very easily detected in the culture medium by western blot.
The antibodies tested are from five different vendors as shown in Table 2: Thermo Fisher Scientific, GeneTex, ABclonal, Bio-Techne and Abcam.
The purpose of this manuscript is to detect the endogenous protein in a relevant sample (culture medium because the protein is secreted). Testing the antibody’s ability to recognize the recombinant proteins is screened internally and routinely by the manufacturer but do not always mean recognition in biologically relevant samples that researchers may be interested in investigating. This study offers a valuable antibody screening in the appropriate sample and in the presence of the most agreed upon type of negative control, a KO.

2.       Human sFRP-1 is a member of the SFRP family. Since many of the antibodies selected are polyclonal in nature, the authors should assess potential cross-reactivity against other closely related family members eg human sFRP-2, sFRP-4 and sFRP-5. Sequence alignment between sFRP-1 against these family members shows stretches of 100% identical amino acids up to 22 amino acids long, portending potential cross-reactivity of anti-sFRP-1 polyclonal antibodies against these entities. Cross-reactivity of the recombinant monoclonal antibodies should also be tested since there is no epitope information for these antibodies.
Cross-reactivity against other closely related family members can only be assessed using other KO controls corresponding to the mentioned genes of interest which we do not have access to. For now, this work is only informative about antibody specificity against sFRP1.

3.       The results for GTX4193, GTX102371, AF1384, AB4193 and AB84003 in Figure 1 and 2 appear contradictory. In Figure 1, these antibodies could not detect sFRP-1 present in concentrated culture media collected from starved A549 cells by western blotting. However, these antibodies were able to detect sFRP-1 at ~35 kDa position in the starting material samples in Figure 2 which are essentially equivalent to the WT samples in Figure 1.
In Figure 2, as indicated in the legend, all starting material and immunoprecipitates corresponding to each antibody tested are western blotted with one antibody, MA5-38193. Since MA5-38193 detects sFRP-1 by WB (Figure 1), signal is visible in all SM lanes.

The detailed antibody characterization platform we have created, along with academic and industry researchers, is available on Protocol Exchange. https://doi.org/10.21203/rs.3.pex-2607/v1
Please see below our response to your concerns, point-by-point. We hope these responses address any misunderstandings or hesitations you previously had, providing a clearer understanding of the manuscripts purpose, the YCharOS initiative as a whole and limitations we face in terms of data interpretation and our protocol.

1.       The authors selected the A549 cell line based on available data on high expression at transcript levels. However, high transcript levels do not necessarily translate to high protein expression. The A549 and A549 sFRP1 KO cell lines as well as some of the antibodies tested were from the same vendor. To have added confidence that the selected antibodies are recognizing endogenous sFRP-1 expressed in A549 cells, the authors should include a recombinant source of sFRP-1 from a different vendor in their western blots. In others words, each antibody is tested against endogenous sFRP-1 expressed in A549 cells, A549 SFRP1 KO (negative control) and recombinant sFRP-1 (eg R&D Systems cat no. 1384-SF).
Agreed, mRNA levels do not always reflect protein expression, however our results show that A549 is an appropriate cell line for antibody testing as the protein was very easily detected in the culture medium by western blot.
The antibodies tested are from five different vendors as shown in Table 2: Thermo Fisher Scientific, GeneTex, ABclonal, Bio-Techne and Abcam.
The purpose of this manuscript is to detect the endogenous protein in a relevant sample (culture medium because the protein is secreted). Testing the antibody’s ability to recognize the recombinant proteins is screened internally and routinely by the manufacturer but do not always mean recognition in biologically relevant samples that researchers may be interested in investigating. This study offers a valuable antibody screening in the appropriate sample and in the presence of the most agreed upon type of negative control, a KO.

2.       Human sFRP-1 is a member of the SFRP family. Since many of the antibodies selected are polyclonal in nature, the authors should assess potential cross-reactivity against other closely related family members eg human sFRP-2, sFRP-4 and sFRP-5. Sequence alignment between sFRP-1 against these family members shows stretches of 100% identical amino acids up to 22 amino acids long, portending potential cross-reactivity of anti-sFRP-1 polyclonal antibodies against these entities. Cross-reactivity of the recombinant monoclonal antibodies should also be tested since there is no epitope information for these antibodies.
Cross-reactivity against other closely related family members can only be assessed using other KO controls corresponding to the mentioned genes of interest which we do not have access to. For now, this work is only informative about antibody specificity against sFRP1.

3.       The results for GTX4193, GTX102371, AF1384, AB4193 and AB84003 in Figure 1 and 2 appear contradictory. In Figure 1, these antibodies could not detect sFRP-1 present in concentrated culture media collected from starved A549 cells by western blotting. However, these antibodies were able to detect sFRP-1 at ~35 kDa position in the starting material samples in Figure 2 which are essentially equivalent to the WT samples in Figure 1.
In Figure 2, as indicated in the legend, all starting material and immunoprecipitates corresponding to each antibody tested are western blotted with one antibody, MA5-38193. Since MA5-38193 detects sFRP-1 by WB (Figure 1), signal is visible in all SM lanes.

The detailed antibody characterization platform we have created, along with academic and industry researchers, is available on Protocol Exchange. https://doi.org/10.21203/rs.3.pex-2607/v1
Competing Interests: No competing interests were disclosed. Close
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Version 1

VERSION 1

PUBLISHED 16 Mar 2023

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Reviewer Report 22 Feb 2024

Oi Wah Liew, Cardiovascular Research Institute, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, Singapore, Singapore

Approved with Reservations

https://doi.org/10.5256/f1000research.143794.r244759

Comments to the manuscript “The identification of high-performing antibodies for Secreted frizzled-related protein 1 (sFRP-1) for use in Western blot and immunoprecipitation”;
Ayoubi R, Southern K, Laflamme C and NeuroSGC/YCharOS collaborative group

GENERAL COMMENT

The manuscript describes the performance of 10 different commercial antibodies (rabbit or goat polyclonal and recombinant monoclonal antibodies) for detecting secreted frizzled-related protein 1 (sFRP-1) for Western blot and immunoprecipitation applications. A high expressing commercial cell line, A549, and its sFRP-1 KO counterpart was used as positive and negative controls for testing.

The work and its findings have scientific merits; however, there are several specific points that should be addressed by the authors. These are provided below:

SPECIFIC COMMENTS
1.       The authors selected the A549 cell line based on available data on high expression at transcript levels. However, high transcript levels do not necessarily translate to high protein expression. The A549 and A549 sFRP1 KO cell lines as well as some of the antibodies tested were from the same vendor. To have added confidence that the selected antibodies are recognizing endogenous sFRP-1 expressed in A549 cells, the authors should include a recombinant source of sFRP-1 from a different vendor in their western blots. In others words, each antibody is tested against endogenous sFRP-1 expressed in A549 cells, A549 SFRP1 KO (negative control) and recombinant sFRP-1 (eg R&D Systems cat no. 1384-SF).
2.       Human sFRP-1 is a member of the SFRP family. Since many of the antibodies selected are polyclonal in nature, the authors should assess potential cross-reactivity against other closely related family members eg human sFRP-2, sFRP-4 and sFRP-5. Sequence alignment between sFRP-1 against these family members shows stretches of 100% identical amino acids up to 22 amino acids long, portending potential cross-reactivity of anti-sFRP-1 polyclonal antibodies against these entities. Cross-reactivity of the recombinant monoclonal antibodies should also be tested since there is no epitope information for these antibodies.
3.       The results for GTX4193, GTX102371, AF1384, AB4193 and AB84003 in Figure 1 and 2 appear contradictory. In Figure 1, these antibodies could not detect sFRP-1 present in concentrated culture media collected from starved A549 cells by western blotting. However, these antibodies were able to detect sFRP-1 at ~35 kDa position in the starting material samples in Figure 2 which are essentially equivalent to the WT samples in Figure 1.

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

Yes
Are the protocols appropriate and is the work technically sound?

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

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

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Biomarkers, antibody generation/characterization and validation, antigen design, recombinant protein expression, genetic engineering, assay development.

CITE

Report a concern

Author Response 13 Apr 2024

Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

13 Apr 2024

Author Response

Thank you Oi Wah Liew for your peer review report!

To address your first comment, we are aware that RNA transcript and protein levels do not always correlate. That ... Continue reading Thank you Oi Wah Liew for your peer review report!

To address your first comment, we are aware that RNA transcript and protein levels do not always correlate. That being said, we have found that an RNA expression value of 2.5 log₂(TPM+1) serves as an appropriate minimal threshold level that will yield detectable protein levels in antibody screening. We understand that this method has its limitations, and in the case that we were unable to detect a strong signal of sFRP-1 in the WT, we have an alternative protocol. A further validation strategy is used to help select the optimal cell line by selecting 4-8 cancer cell lines with high RNA scores to assess and compare protein expression in cell lines in WB. Although outside the scope of our standardized protocol, we would recommend to researchers to use this study as a guide to select high-quality antibodies, then validate the antibodies using a commercial recombinant sFRP-1, as suggested.

The evaluation of cross reactivity of the sFRP-1 antibodies is, once again, outside the scope of this study. We leave these subsequent studies up to the experts studying the sFRP family of proteins. This study is part of a broader collaborative initiative aimed at validating commercial antibodies for every human protein, preventing us from performing follow up studies for every protein target.

To address your final comment, in Western blot, 50 μg of sample was processed onto the gel l whereas in Immunoprecipitation (IP), 100 μg of protein sample was processed. This data is found the Figure legends. This can explain the antibodies ability to detect a more concentrated amount of proteins and generate a signal in the starting material in Figure 2 as compared to Figure 1. Regardless, a successful antibody should enrich its target in the IP compared to the starting material, and deplete it from the unbound fraction, as seen for antibody MA5-32675**.

For more information on how to interpret the antibody characterization data in this study, an editorial by Biddle et al., featured on our YCharOS gateway, can serve as an excellent guide. https://f1000research.com/articles/12-1344

To address comments from another reviewer’s report, a second version of this Data Note has been submitted to F1000. We encourage you to review this subsequent version as well as we believe it will help address some of your comments and provide a clearer definition of the YCharOS initiative.
Thank you Oi Wah Liew for your peer review report!

To address your first comment, we are aware that RNA transcript and protein levels do not always correlate. That being said, we have found that an RNA expression value of 2.5 log₂(TPM+1) serves as an appropriate minimal threshold level that will yield detectable protein levels in antibody screening. We understand that this method has its limitations, and in the case that we were unable to detect a strong signal of sFRP-1 in the WT, we have an alternative protocol. A further validation strategy is used to help select the optimal cell line by selecting 4-8 cancer cell lines with high RNA scores to assess and compare protein expression in cell lines in WB. Although outside the scope of our standardized protocol, we would recommend to researchers to use this study as a guide to select high-quality antibodies, then validate the antibodies using a commercial recombinant sFRP-1, as suggested.

The evaluation of cross reactivity of the sFRP-1 antibodies is, once again, outside the scope of this study. We leave these subsequent studies up to the experts studying the sFRP family of proteins. This study is part of a broader collaborative initiative aimed at validating commercial antibodies for every human protein, preventing us from performing follow up studies for every protein target.

To address your final comment, in Western blot, 50 μg of sample was processed onto the gel l whereas in Immunoprecipitation (IP), 100 μg of protein sample was processed. This data is found the Figure legends. This can explain the antibodies ability to detect a more concentrated amount of proteins and generate a signal in the starting material in Figure 2 as compared to Figure 1. Regardless, a successful antibody should enrich its target in the IP compared to the starting material, and deplete it from the unbound fraction, as seen for antibody MA5-32675**.

For more information on how to interpret the antibody characterization data in this study, an editorial by Biddle et al., featured on our YCharOS gateway, can serve as an excellent guide. https://f1000research.com/articles/12-1344

To address comments from another reviewer’s report, a second version of this Data Note has been submitted to F1000. We encourage you to review this subsequent version as well as we believe it will help address some of your comments and provide a clearer definition of the YCharOS initiative.
Competing Interests: No competing interests were disclosed. Close
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Respond or Comment

COMMENTS ON THIS REPORT

Author Response 13 Apr 2024

Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

13 Apr 2024

Author Response

Thank you Oi Wah Liew for your peer review report!

To address your first comment, we are aware that RNA transcript and protein levels do not always correlate. That ... Continue reading Thank you Oi Wah Liew for your peer review report!

To address your first comment, we are aware that RNA transcript and protein levels do not always correlate. That being said, we have found that an RNA expression value of 2.5 log₂(TPM+1) serves as an appropriate minimal threshold level that will yield detectable protein levels in antibody screening. We understand that this method has its limitations, and in the case that we were unable to detect a strong signal of sFRP-1 in the WT, we have an alternative protocol. A further validation strategy is used to help select the optimal cell line by selecting 4-8 cancer cell lines with high RNA scores to assess and compare protein expression in cell lines in WB. Although outside the scope of our standardized protocol, we would recommend to researchers to use this study as a guide to select high-quality antibodies, then validate the antibodies using a commercial recombinant sFRP-1, as suggested.

The evaluation of cross reactivity of the sFRP-1 antibodies is, once again, outside the scope of this study. We leave these subsequent studies up to the experts studying the sFRP family of proteins. This study is part of a broader collaborative initiative aimed at validating commercial antibodies for every human protein, preventing us from performing follow up studies for every protein target.

To address your final comment, in Western blot, 50 μg of sample was processed onto the gel l whereas in Immunoprecipitation (IP), 100 μg of protein sample was processed. This data is found the Figure legends. This can explain the antibodies ability to detect a more concentrated amount of proteins and generate a signal in the starting material in Figure 2 as compared to Figure 1. Regardless, a successful antibody should enrich its target in the IP compared to the starting material, and deplete it from the unbound fraction, as seen for antibody MA5-32675**.

For more information on how to interpret the antibody characterization data in this study, an editorial by Biddle et al., featured on our YCharOS gateway, can serve as an excellent guide. https://f1000research.com/articles/12-1344

To address comments from another reviewer’s report, a second version of this Data Note has been submitted to F1000. We encourage you to review this subsequent version as well as we believe it will help address some of your comments and provide a clearer definition of the YCharOS initiative.
Thank you Oi Wah Liew for your peer review report!

To address your first comment, we are aware that RNA transcript and protein levels do not always correlate. That being said, we have found that an RNA expression value of 2.5 log₂(TPM+1) serves as an appropriate minimal threshold level that will yield detectable protein levels in antibody screening. We understand that this method has its limitations, and in the case that we were unable to detect a strong signal of sFRP-1 in the WT, we have an alternative protocol. A further validation strategy is used to help select the optimal cell line by selecting 4-8 cancer cell lines with high RNA scores to assess and compare protein expression in cell lines in WB. Although outside the scope of our standardized protocol, we would recommend to researchers to use this study as a guide to select high-quality antibodies, then validate the antibodies using a commercial recombinant sFRP-1, as suggested.

The evaluation of cross reactivity of the sFRP-1 antibodies is, once again, outside the scope of this study. We leave these subsequent studies up to the experts studying the sFRP family of proteins. This study is part of a broader collaborative initiative aimed at validating commercial antibodies for every human protein, preventing us from performing follow up studies for every protein target.

To address your final comment, in Western blot, 50 μg of sample was processed onto the gel l whereas in Immunoprecipitation (IP), 100 μg of protein sample was processed. This data is found the Figure legends. This can explain the antibodies ability to detect a more concentrated amount of proteins and generate a signal in the starting material in Figure 2 as compared to Figure 1. Regardless, a successful antibody should enrich its target in the IP compared to the starting material, and deplete it from the unbound fraction, as seen for antibody MA5-32675**.

For more information on how to interpret the antibody characterization data in this study, an editorial by Biddle et al., featured on our YCharOS gateway, can serve as an excellent guide. https://f1000research.com/articles/12-1344

To address comments from another reviewer’s report, a second version of this Data Note has been submitted to F1000. We encourage you to review this subsequent version as well as we believe it will help address some of your comments and provide a clearer definition of the YCharOS initiative.
Competing Interests: No competing interests were disclosed. Close
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Views

Reviewer Report 08 Feb 2024

Viyatprajna Acharya, KIMS, KIIT, Bhubaneswar, India

Approved with Reservations

https://doi.org/10.5256/f1000research.143794.r185117

The result can be discussed a little more elaborately. Methods are well-explained. Supporting diagram for immunoprecipitation and Western blot may be provided. The conclusion can be more explicit such as describing how many samples were actually suitable or better over ... Continue reading

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

Yes
Are the protocols appropriate and is the work technically sound?

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

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

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Immunochemistry, metabolomics, cancer, oxidative stress.

CITE

Report a concern

Author Response 13 Apr 2024

Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

13 Apr 2024

Author Response

Dear Viyatprajna Acharya,
Thank you for submitting a peer review report for this Data Note.

An open-access Dataset is available under the “Data Availability” section. You can find the ... Continue reading Dear Viyatprajna Acharya,
Thank you for submitting a peer review report for this Data Note.

An open-access Dataset is available under the “Data Availability” section. You can find the supporting diagrams and underlying data for Western blot and immunoprecipitation experiments within this dataset. As an organization that prioritizes open science, we ensure that all data is transparent and readily available which is why all of our publications have equivalent Datasets.

The objective of this article is not to interpret the results nor score the performance of the antibodies. Given that it is formatted as a Data Note, it does not require the results to be discussed or concluded. YCharOS is a public-private organization whose mission is to characterize antibodies for every human protein and deliver the research as a collective good for the scientific community. That being said, we understand how this intention may be misinterpreted. A new version has been submitted, to include modifications to the title, introduction and results&discussion section that ensure our goals clearly are aligned and defined to all viewers.

References 8-11 have been edited and will be complete in the newly submitted version.

Thank you for your feedback and corrections!
Dear Viyatprajna Acharya,
Thank you for submitting a peer review report for this Data Note.

An open-access Dataset is available under the “Data Availability” section. You can find the supporting diagrams and underlying data for Western blot and immunoprecipitation experiments within this dataset. As an organization that prioritizes open science, we ensure that all data is transparent and readily available which is why all of our publications have equivalent Datasets.

The objective of this article is not to interpret the results nor score the performance of the antibodies. Given that it is formatted as a Data Note, it does not require the results to be discussed or concluded. YCharOS is a public-private organization whose mission is to characterize antibodies for every human protein and deliver the research as a collective good for the scientific community. That being said, we understand how this intention may be misinterpreted. A new version has been submitted, to include modifications to the title, introduction and results&discussion section that ensure our goals clearly are aligned and defined to all viewers.

References 8-11 have been edited and will be complete in the newly submitted version.

Thank you for your feedback and corrections!
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 13 Apr 2024

Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

13 Apr 2024

Author Response

Dear Viyatprajna Acharya,
Thank you for submitting a peer review report for this Data Note.

An open-access Dataset is available under the “Data Availability” section. You can find the ... Continue reading Dear Viyatprajna Acharya,
Thank you for submitting a peer review report for this Data Note.

An open-access Dataset is available under the “Data Availability” section. You can find the supporting diagrams and underlying data for Western blot and immunoprecipitation experiments within this dataset. As an organization that prioritizes open science, we ensure that all data is transparent and readily available which is why all of our publications have equivalent Datasets.

The objective of this article is not to interpret the results nor score the performance of the antibodies. Given that it is formatted as a Data Note, it does not require the results to be discussed or concluded. YCharOS is a public-private organization whose mission is to characterize antibodies for every human protein and deliver the research as a collective good for the scientific community. That being said, we understand how this intention may be misinterpreted. A new version has been submitted, to include modifications to the title, introduction and results&discussion section that ensure our goals clearly are aligned and defined to all viewers.

References 8-11 have been edited and will be complete in the newly submitted version.

Thank you for your feedback and corrections!
Dear Viyatprajna Acharya,
Thank you for submitting a peer review report for this Data Note.

An open-access Dataset is available under the “Data Availability” section. You can find the supporting diagrams and underlying data for Western blot and immunoprecipitation experiments within this dataset. As an organization that prioritizes open science, we ensure that all data is transparent and readily available which is why all of our publications have equivalent Datasets.

The objective of this article is not to interpret the results nor score the performance of the antibodies. Given that it is formatted as a Data Note, it does not require the results to be discussed or concluded. YCharOS is a public-private organization whose mission is to characterize antibodies for every human protein and deliver the research as a collective good for the scientific community. That being said, we understand how this intention may be misinterpreted. A new version has been submitted, to include modifications to the title, introduction and results&discussion section that ensure our goals clearly are aligned and defined to all viewers.

References 8-11 have been edited and will be complete in the newly submitted version.

Thank you for your feedback and corrections!
Competing Interests: No competing interests were disclosed. Close
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Version 2

VERSION 2 PUBLISHED 16 Mar 2023

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Viyatprajna Acharya, KIMS, KIIT, Bhubaneswar, India
Oi Wah Liew, National University of Singapore, National University Health System, Singapore, Singapore
Lynne Howells, University of Leicester, Leicester, UK

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23 Sep 2024 | for Version 2

Lynne Howells, University of Leicester, Leicester, UK

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Approved

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

Yes
Are the protocols appropriate and is the work technically sound?

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

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

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Cancer biology, cell signalling (WB, IP, FACS, IHC, IF)

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.

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18 Apr 2024 | for Version 2

Oi Wah Liew, Cardiovascular Research Institute, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, Singapore, Singapore

15 Views Cite this report Responses(1)

Approved With Reservations

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Biomarkers, antibody generation/characterization and validation, antigen design, recombinant protein expression, genetic engineering, assay development.

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Author Response

28 Jun 2024

Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

Please see below our response to your concerns, point-by-point. We hope these responses address any misunderstandings or hesitations you previously had, providing a clearer understanding of the manuscripts purpose, the YCharOS initiative as a whole and limitations we face in terms of data interpretation and our protocol.

1.       The authors selected the A549 cell line based on available data on high expression at transcript levels. However, high transcript levels do not necessarily translate to high protein expression. The A549 and A549 sFRP1 KO cell lines as well as some of the antibodies tested were from the same vendor. To have added confidence that the selected antibodies are recognizing endogenous sFRP-1 expressed in A549 cells, the authors should include a recombinant source of sFRP-1 from a different vendor in their western blots. In others words, each antibody is tested against endogenous sFRP-1 expressed in A549 cells, A549 SFRP1 KO (negative control) and recombinant sFRP-1 (eg R&D Systems cat no. 1384-SF).
Agreed, mRNA levels do not always reflect protein expression, however our results show that A549 is an appropriate cell line for antibody testing as the protein was very easily detected in the culture medium by western blot.
The antibodies tested are from five different vendors as shown in Table 2: Thermo Fisher Scientific, GeneTex, ABclonal, Bio-Techne and Abcam.
The purpose of this manuscript is to detect the endogenous protein in a relevant sample (culture medium because the protein is secreted). Testing the antibody’s ability to recognize the recombinant proteins is screened internally and routinely by the manufacturer but do not always mean recognition in biologically relevant samples that researchers may be interested in investigating. This study offers a valuable antibody screening in the appropriate sample and in the presence of the most agreed upon type of negative control, a KO.

2.       Human sFRP-1 is a member of the SFRP family. Since many of the antibodies selected are polyclonal in nature, the authors should assess potential cross-reactivity against other closely related family members eg human sFRP-2, sFRP-4 and sFRP-5. Sequence alignment between sFRP-1 against these family members shows stretches of 100% identical amino acids up to 22 amino acids long, portending potential cross-reactivity of anti-sFRP-1 polyclonal antibodies against these entities. Cross-reactivity of the recombinant monoclonal antibodies should also be tested since there is no epitope information for these antibodies.
Cross-reactivity against other closely related family members can only be assessed using other KO controls corresponding to the mentioned genes of interest which we do not have access to. For now, this work is only informative about antibody specificity against sFRP1.

3.       The results for GTX4193, GTX102371, AF1384, AB4193 and AB84003 in Figure 1 and 2 appear contradictory. In Figure 1, these antibodies could not detect sFRP-1 present in concentrated culture media collected from starved A549 cells by western blotting. However, these antibodies were able to detect sFRP-1 at ~35 kDa position in the starting material samples in Figure 2 which are essentially equivalent to the WT samples in Figure 1.
In Figure 2, as indicated in the legend, all starting material and immunoprecipitates corresponding to each antibody tested are western blotted with one antibody, MA5-38193. Since MA5-38193 detects sFRP-1 by WB (Figure 1), signal is visible in all SM lanes.

The detailed antibody characterization platform we have created, along with academic and industry researchers, is available on Protocol Exchange. https://doi.org/10.21203/rs.3.pex-2607/v1

View more View less

Competing Interests

No competing interests were disclosed.

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22 Feb 2024 | for Version 1

Oi Wah Liew, Cardiovascular Research Institute, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, Singapore, Singapore

21 Views Cite this report Responses(1)

Approved With Reservations

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

Yes
Are the protocols appropriate and is the work technically sound?

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

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

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Biomarkers, antibody generation/characterization and validation, antigen design, recombinant protein expression, genetic engineering, assay development.

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Responses (1)

Author Response

13 Apr 2024

Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

Thank you Oi Wah Liew for your peer review report!

To address your first comment, we are aware that RNA transcript and protein levels do not always correlate. That being said, we have found that an RNA expression value of 2.5 log₂(TPM+1) serves as an appropriate minimal threshold level that will yield detectable protein levels in antibody screening. We understand that this method has its limitations, and in the case that we were unable to detect a strong signal of sFRP-1 in the WT, we have an alternative protocol. A further validation strategy is used to help select the optimal cell line by selecting 4-8 cancer cell lines with high RNA scores to assess and compare protein expression in cell lines in WB. Although outside the scope of our standardized protocol, we would recommend to researchers to use this study as a guide to select high-quality antibodies, then validate the antibodies using a commercial recombinant sFRP-1, as suggested.

The evaluation of cross reactivity of the sFRP-1 antibodies is, once again, outside the scope of this study. We leave these subsequent studies up to the experts studying the sFRP family of proteins. This study is part of a broader collaborative initiative aimed at validating commercial antibodies for every human protein, preventing us from performing follow up studies for every protein target.

To address your final comment, in Western blot, 50 μg of sample was processed onto the gel l whereas in Immunoprecipitation (IP), 100 μg of protein sample was processed. This data is found the Figure legends. This can explain the antibodies ability to detect a more concentrated amount of proteins and generate a signal in the starting material in Figure 2 as compared to Figure 1. Regardless, a successful antibody should enrich its target in the IP compared to the starting material, and deplete it from the unbound fraction, as seen for antibody MA5-32675**.

For more information on how to interpret the antibody characterization data in this study, an editorial by Biddle et al., featured on our YCharOS gateway, can serve as an excellent guide. https://f1000research.com/articles/12-1344

To address comments from another reviewer’s report, a second version of this Data Note has been submitted to F1000. We encourage you to review this subsequent version as well as we believe it will help address some of your comments and provide a clearer definition of the YCharOS initiative.

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Competing Interests

No competing interests were disclosed.

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Reviewer Report

24 Views

08 Feb 2024 | for Version 1

Viyatprajna Acharya, KIMS, KIIT, Bhubaneswar, India

24 Views Cite this report Responses(1)

Approved With Reservations

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

Yes
Are the protocols appropriate and is the work technically sound?

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

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

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Immunochemistry, metabolomics, cancer, oxidative stress.

Respond to this report

Responses (1)

Author Response

13 Apr 2024

Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

Dear Viyatprajna Acharya,
Thank you for submitting a peer review report for this Data Note.

An open-access Dataset is available under the “Data Availability” section. You can find the supporting diagrams and underlying data for Western blot and immunoprecipitation experiments within this dataset. As an organization that prioritizes open science, we ensure that all data is transparent and readily available which is why all of our publications have equivalent Datasets.

The objective of this article is not to interpret the results nor score the performance of the antibodies. Given that it is formatted as a Data Note, it does not require the results to be discussed or concluded. YCharOS is a public-private organization whose mission is to characterize antibodies for every human protein and deliver the research as a collective good for the scientific community. That being said, we understand how this intention may be misinterpreted. A new version has been submitted, to include modifications to the title, introduction and results&discussion section that ensure our goals clearly are aligned and defined to all viewers.

References 8-11 have been edited and will be complete in the newly submitted version.

Thank you for your feedback and corrections!

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Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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[7] 7. Licciardello MP, Workman P: The era of high-quality chemical probes. RSC Med. Chem. 2022; 13(12): 1446–1459. PubMed Abstract | Publisher Full Text | Free Full Text

[8] 8. Carter AJ, et al.: Target 2035: probing the human proteome. Drug Discov. Today. 2019; 24(11): 2111–2115. PubMed Abstract | Publisher Full Text

[9] 9. 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. Publisher Full Text

[10] 10. Alshafie W, Fotouhi M, Shlaifer I, et al.: Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence. F1000Res. 2022; 11: 977. Publisher Full Text

[11] 11. Ayoubi R, McPherson PS, Laflamme C: Antibody Screening by Immunoblot.2021. Publisher Full Text

[12] 12. Ayoubi R, Fotouhi M, McPherson P, et al.: Antibody screening by Immunoprecitation.2021. Publisher Full Text

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A guide to selecting high-performing antibodies for Secreted frizzled-related protein 1 (sFRP-1) for use in Western Blot and immunoprecipitation

Abstract

Keywords

Revised Amendments from Version 1

Introduction

Results and discussion

Table 1. Summary of the cell lines used.

Table 2. Summary of the Secreted frizzled-related protein 1 antibodies tested.

Figure 1. Secreted frizzled-related protein 1 antibody screening by Western Blot on culture media.

Figure 2. Secreted frizzled-related protein 1 antibody screening by immunoprecipitation on culture media.

Methods

Antibodies

Cell culture

Antibody screening by Western Blot on culture media

Antibody screening by immunoprecipitation on culture media

Data availability

Underlying data

Acknowledgments

References

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