A guide to selecting high-performing antibodies for human Midkine for use in Western blot and immunoprecipitation

Riham Ayoubi; Kathleen Southern; Carl Laflamme; NeuroSGC/YCharOS Collaborative Group

doi:10.12688/f1000research.130587.4

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Revised

A guide to selecting high-performing antibodies for human Midkine for use in Western blot and immunoprecipitation

[version 4; peer review: 2 approved]

Previously titled: The identification of high-performing antibodies for Midkine for use in Western blot and immunoprecipitation

Riham Ayoubi¹, Kathleen Southern¹, Carl Laflamme ¹, NeuroSGC/YCharOS Collaborative Group

PUBLISHED 01 Aug 2024

Author details Author details

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

Riham Ayoubi
Roles: Investigation, Writing – Original Draft Preparation

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 – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

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

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

Abstract

Midkine is a secreted protein that acts as a growth factor or cytokine involved in cell survival and inflammatory processes. It accumulates in amyloid plaques, which are hallmarks of Alzheimer’s Disease (AD). The reproducibility of Midkine research would be enhanced if the community had access to well-characterized anti-Midkine antibodies. In this study, we characterized 8 commercial Midkine antibodies for Western blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in a knockout cell line and isogenic parental control. 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 #P21741, MDK, Midkine, antibody validation, antibody characterization, 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) and by the Government of Canada through Genome Canada and Ontario Genomics (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 (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. 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.
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 human Midkine for use in Western blot and immunoprecipitation [version 4; peer review: 2 approved]. F1000Research 2024, 12:148 (https://doi.org/10.12688/f1000research.130587.4) First published: 09 Feb 2023, 12:148 (https://doi.org/10.12688/f1000research.130587.1) Latest published: 01 Aug 2024, 12:148 (https://doi.org/10.12688/f1000research.130587.4)

Revised Amendments from Version 3

In our efforts to ensure reproducibility, version 4 includes amendments to the Methods and Figure 1 legend to provide detailed information on how the HAP1 WT and MDK KO cells were prepared prior to Western blot evaluation.

See the authors' detailed response to the review by Jean-Pierre Bellier
See the authors' detailed response to the review by Jens-Ulrich Rahfeld

Introduction

The neurotrophic and developmental factor Midkine is a secreted heparin-binding cytokine.¹ It mediates its diverse physiological functions by binding to cell-surface proteoglycan receptors via their chondroitin sulfate groups, thereby regulating cell proliferation, migration and differentiation.²^–⁴

Midkine is involved in numerous processes that promote cell growth, and may contribute to the pathogenesis of inflammatory diseases.¹^,⁴ Proteomic analyses have found that Midkine is highly enriched in amyloid-beta plaques, indicating its potential as a biomarker and/or therapeutic target for Alzheimer’s Disease (AD).⁵^–⁷ Mechanistic studies would be greatly facilitated by the availability of high-quality antibodies for Midkine.

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.⁸^–¹⁰ Here, we compared the performance of a range of commercially available antibodies for Midkine 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 Midkine properties and function.

Results and discussion

Our standard protocol involves comparing readouts from wild-type and knockout cells.¹¹^–¹³ The first step is to identify a human 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 human HAP1 cells expressed the Midkine transcript at RNA levels above the average range of cancer cells analyzed.¹⁴ Parental and MDK knockout human HAP1 cells were obtained from Horizon Discovery (Table 1).

Table 1. Summary of the cell lines used.

Institution	Catalog number	RRID (Cellosaurus)	Cell line	Genotype
Horizon Discovery	C631	CVCL_Y019	HAP1	WT
Horizon Discovery	HZGHC007906c008	CVCL_C6Y2	HAP1	MDK KO

Midkine is predicted to be a secreted protein. Accordingly, we collected concentrated culture media from both wild-type and MDK 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 the tested antibodies are shown in Figures 1 and 2.

Table 2. Summary of the Midkine antibodies tested.

Company	Catalog number	Lot number	RRID (Antibody Registry)	Clonality	Clone ID	Host	Concentration (μg/μl)	Initial vendors recommended applications
GeneTex	GTX108439	39855	AB_1950903	polyclonal	-	rabbit	0.33	Wb
GeneTex	GTX116089^a	40597	AB_11165850	polyclonal	-	rabbit	1.00	Wb
Bio-Techne	AF-258-PB	WE0519091	AB_2143400	polyclonal	-	goat	0.20	Wb
Bio-Techne	MAB2582**	CMHR0219071	AB_2893288	recombinant-mono	1011522	mouse	0.50	IF
Bio-Techne	MAB2583*	CMLT0120031	AB_2893289	monoclonal	1011622	mouse	0.50	-
Bio-Techne	NBP2-66948**^a	HN0907	AB_2893290	recombinant-mono	JF096-5	rabbit	1.00	Wb, IP
Thermo	MA5-32538**	WD3265256	AB_2809815	recombinant-mono	JF096-5	rabbit	1.00	Wb
Abcam	ab52637**	GR3315059-2	AB_880698	recombinant-mono	EP1143Y	rabbit	0.10	Wb, IP, IF

* Monoclonal antibody.

** Recombinant antibody.

a Antibodies that have been discontinued subsequent to the release of this study.

Figure 1. Midkine antibody screening by Western blot on culture media.

A) Schematic of the protocol used to concentrate the culture media. B) Concentrated media of HAP1 (WT and MDK KO) were prepared, and ~30 μg of protein was processed for Western blot with the indicated Midkine antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. An exceptions was given to antibody GTX116089, which was titrated to 1/1000, as the signal was too weak when following the supplier’s recommendations. When suppliers did not recommend dilutions, we tested the antibodies at 1/200. Antibody dilution used: GTX108439 at 1/1000; GTX116089 at 1/100; AF-258-PB at 1/100; MAB2582** at 1/200; MAB2583* at 1/200; NBP2-66948** at 1/500; MA5-32538** at 1/500; ab52637** at 1/500. Predicted band size: 15 kDa. *Monoclonal antibody, **Recombinant antibody.

Figure 2. Midkine antibody screening by immunoprecipitation on culture media.

Concentrated culture media were prepared as in 1A). Immunoprecipitation was performed using 1.0 μg of the indicated Midkine antibodies pre-coupled to either protein A or protein G magnetic beads. Samples were washed and processed for Western blot with the indicated Midkine antibody. For Western blot, AF-258-PB was used at 1/500, NBP2-66948** at 1/500, MA5-32538** at 1/200 and ab52637** at 1/200. The Ponceau stained transfers of each blot are shown. SM = 10% starting material; UB = 10% unbound fraction; IP = immunoprecipitate; HC = antibody heavy chain. *Monoclonal antibody, **Recombinant antibody.

In conclusion, we screened eight Midkine commercial antibodies by Western blot and immunoprecipitation. Several high-quality antibodies that successfully detect Midkine under our standardized experimental conditions can be identified. 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.

Antibodies GTX 116089 and NBP2-66948 were removed from the commercial antibody market and are no longer available. Antibody MA5-32538 has the same clone ID as NBP2-66948, and is still available on the market as an alternative.

Methods

Antibodies

All Midkine antibodies are listed in Table 2. Peroxidase-conjugated goat anti-rabbit, anti-mouse and donkey anti-goat antibodies are from Thermo Fisher Scientific (cat. number 65-6120, 62-6520 and A15999, respectively).

Cell culture

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 medium

HAP1 cells (WT and MDK KO) were grown in 20 ml complete medium to 80% confluence in 150 mm dishes. At 80% confluency, HAP1 cells were at a density of ~1.0 × 10⁶/ml (~20.0 × 10⁶/20 ml). To prepare for protein extraction, cells 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 initially concentrated using Amicon Ultra-15 Centrifugal Filter Units (MilliporeSigma cat. number UFC9010) by centrifuging at 4000 × g for 15 min. The resulting 500 μl of concentrated media from each filter unit were centrifuged again at 4000 × g for 15 min using Amicon Ultra- 0.5 Centrifugal Filter Units (MilliporeSigma cat. number UFC5010) to 200 μl. The final protein concentration was 0.3 mg/ml and 100 μl (30 μg) of media were used for each blot.

Western blots were performed as described in our standard operating procedure.¹³^,¹⁵ A Bradford reagent assay was performed to determine the protein concentration of the culture medium. The concentration was then adjusted to ensure equal amounts of protein were loaded in each gel. Western blots were performed with large 10-20% gradient polyacrylamide gels, loaded with 30 μg of protein in each lane and then 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 medium

Immunoprecipitation was performed as described in our standard operating procedure.¹³^,¹⁶ Antibody-bead conjugates were prepared by adding 1.0 μg of antibody to 500 μl of Pierce IP Lysis from Thermo Fisher Scientific (cat. number 87788) in a microcentrifuge tube, together with 30 μl of Dynabeads protein A - (for rabbit antibodies) or protein G - (for mouse and goat antibodies) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively). Tubes were rocked overnight at 4°C followed by two washes with the Pierce IP Buffer to remove unbound antibodies. 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 1× Halt Protease and Phosphatase Inhibitor Cocktail from Thermo Fisher Scientific (cat. number 78446).

Starved HAP1 WT culture media were concentrated as described above. The concentrated culture media were diluted in Pierce IP Lysis Buffer, and 1ml aliquots at 0.3 mg/ml of lysate were incubated with an antibody-bead conjugate for ~2 hrs 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 immunoblot on 10-20% polyacrylamide gels. Prot-A: HRP was used as a secondary detection system (MilliporeSigma, cat. number P8651) at a dilution of 0.4 μg/ml for an experiment where a rabbit antibody was used for both immunoprecipitation and its corresponding Western Blot.

Data availability

Underlying data

Zenodo: Antibody Characterization Report for Midkine, https://doi.org/10.5281/zenodo.5644321.¹⁷

Zenodo: Dataset for the Midkine antibody screening study, https://doi.org/10.5281/zenodo.7530472.¹⁸

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

Acknowledgment

The authors would like to thank the NeuroSGC/YCharOS collaborative group for 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 this group can be found below. NeuroSGC/YCharOS collaborative group: Riham Ayoubi, Kathleen Southern, Peter S. McPherson, Aled M. Edwards, Chetan Raina and Carl Laflamme.

An earlier version of this article can be found on Zenodo (doi:10.5281/zenodo.5644321).¹⁷

References

1. Muramatsu T: Structure and function of midkine as the basis of its pharmacological effects. Br. J. Pharmacol. 2014; 171(4): 814–826. PubMed Abstract | Publisher Full Text | Free Full Text
2. Kurosawa N, Chen GY, Kadomatsu K, et al.: Glypican-2 binds to midkine: the role of glypican-2 in neuronal cell adhesion and neurite outgrowth. Glycoconj. J. 2001; 18(6): 499–507. Publisher Full Text
3. Maeda N, Ichihara-Tanaka K, Kimura T, et al.: A receptor-like protein-tyrosine phosphatase PTPzeta/RPTPbeta binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPzeta. J. Biol. Chem. 1999; 274(18): 12474–12479. Publisher Full Text
4. Ross-Munro E, Kwa F, Kreiner J, et al.: Midkine: The Who, What, Where, and When of a Promising Neurotrophic Therapy for Perinatal Brain Injury. Front. Neurol. 2020; 11: 568814. PubMed Abstract | Publisher Full Text | Free Full Text
5. Drummond E, Kavanagh T, Pires G, et al.: The amyloid plaque proteome in early onset Alzheimer's disease and Down syndrome. Acta Neuropathol. Commun. 2022; 10(1): 53. PubMed Abstract | Publisher Full Text | Free Full Text
6. 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
7. Yasuhara O, Muramatsu H, Kim SU, et al.: Midkine, a novel neurotrophic factor, is present in senile plaques of Alzheimer disease. Biochem. Biophys. Res. Commun. 1993; 192(1): 246–251. PubMed Abstract | Publisher Full Text
8. 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. PubMed Abstract | Publisher Full Text | Free Full Text
9. 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
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11. 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
12. 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
13. Ayoubi R, Ryan J, Bolivar SG, et al.: A consensus platform for antibody characterization (Version 1). Protocol Exchange. 2024. Publisher Full Text
14. Ghandi M, Huang FW, Jane-Valbuena J, et al.: Next-generation characterization of the Cancer Cell Line Encyclopedia. Nature. 2019; 569(7757): 503–508. PubMed Abstract | Publisher Full Text | Free Full Text
15. Ayoubi R, McPherson PS, Laflamme C: Antibody Screening by Immunoblot.2021. Publisher Full Text
16. Ayoubi R, Fotouhi M, McPherson P, et al.: Antibody screening by Immunoprecitation.2021. Publisher Full Text
17. Ayoubi R, McPherson PS, Laflamme C: Antibody Characterization Report for Midkine.2021. Publisher Full Text
18. Laflamme C: Dataset for the Midkine antibody screening study. [Data set]. Zenodo. 2023. Publisher Full Text

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Version 4

VERSION 4 PUBLISHED 09 Feb 2023

Author details Author details

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

Riham Ayoubi
Roles: Investigation, Writing – Original Draft Preparation

Kathleen Southern
Roles: Writing – Original Draft Preparation, 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) and by the Government of Canada through Genome Canada and Ontario Genomics (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 (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. 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.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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© 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 human Midkine for use in Western blot and immunoprecipitation [version 4; peer review: 2 approved]. F1000Research 2024, 12:148 (https://doi.org/10.12688/f1000research.130587.4)

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Reviewer Report 31 Jul 2024

Jens-Ulrich Rahfeld, Fraunhofer Institute for Cell Therapy and Immunology, Department Molecular Drug Biochemistry and Therapy, Weinbergweg 22, 06120, Halle, Germany

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Reviewer Report 13 May 2024

Jens-Ulrich Rahfeld, Fraunhofer Institute for Cell Therapy and Immunology, Department Molecular Drug Biochemistry and Therapy, Weinbergweg 22, 06120, Halle, Germany

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https://doi.org/10.5256/f1000research.160165.r269176

The results of the publication are very helpful for the selection of suitable Midkine antibodies.

However, there are ambiguities in the experimental and methodological part:
The protocol mentions that the cell culture supernatants were obtained from starving cultures and then concentrated (also shown in scheme A in Figure 1.
For the reproducibility of the preparation of these supernatants, there is no information on the cell density/ml of the cultivated HAP1 cells or the initial volume of the cell culture supernatant concentrated for analysis.

Furthermore, the description of Figure 1 B refers to cell lysates:
" Lysates of HAP1 (WT and MDK KO) were prepared, and ~30 μg of protein was processed for Western blot."
This is not explained in the experimental section. This should definitely be explained or corrected.

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?

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

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: proteinchemisty

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 23 Jul 2024

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

23 Jul 2024

Author Response

Thank you to Dr. Jens-Ulrich Rahfeld for your comprehensive review of this article which presents as a guide to selecting high-performing antibodies for Midkine to progress research of this target ... Continue reading Thank you to Dr. Jens-Ulrich Rahfeld for your comprehensive review of this article which presents as a guide to selecting high-performing antibodies for Midkine to progress research of this target associated to many pathological conditions. We hope our response to your comments (italicized) successfully answers any concerns you previously had.

Reviewer Comment:
However, there are ambiguities in the experimental and methodological part:
The protocol mentions that the cell culture supernatants were obtained from starving cultures and then concentrated (also shown in scheme A in Figure 1.
For the reproducibility of the preparation of these supernatants, there is no information on the cell density/ml of the cultivated HAP1 cells or the initial volume of the cell culture supernatant concentrated for analysis.

Author Response:
To facilitate reproducibility of the antibody characterization platform utilized by the authors, we have included a reference to the full procedure, available on Protocol exchange (https://doi.org/10.21203/rs.3.pex-2607/v1), to the new version (Version 3) of the article that will be submitted shortly. That being said, once the concentrated cell media is collected, the protein concentration is determined using Bradford reagent protein assay, and the concentrations are adjusted to ensure equal protein amounts are loaded into the gel. We understand the authors were not clear on this aspect of the sample preparation procedure for western blot and have included this to the Methods section in the new version of the article.

Reviewer Comment:
Furthermore, the description of Figure 1 B refers to cell lysates:
" Lysates of HAP1 (WT and MDK KO) were prepared, and ~30 μg of protein was processed for Western blot."
This is not explained in the experimental section. This should definitely be explained or corrected.

Author Response:
To the “Antibody screening by Western blot on culture media” area of the methods section, we have clarified that 30 μg of protein was added to each lane of the polyacrylamide gel. We’ve also referenced our full detailed procedure, which includes troubleshooting when unprecedented results or issues arise.
Thank you to Dr. Jens-Ulrich Rahfeld for your comprehensive review of this article which presents as a guide to selecting high-performing antibodies for Midkine to progress research of this target associated to many pathological conditions. We hope our response to your comments (italicized) successfully answers any concerns you previously had.

Reviewer Comment:
However, there are ambiguities in the experimental and methodological part:
The protocol mentions that the cell culture supernatants were obtained from starving cultures and then concentrated (also shown in scheme A in Figure 1.
For the reproducibility of the preparation of these supernatants, there is no information on the cell density/ml of the cultivated HAP1 cells or the initial volume of the cell culture supernatant concentrated for analysis.

Author Response:
To facilitate reproducibility of the antibody characterization platform utilized by the authors, we have included a reference to the full procedure, available on Protocol exchange (https://doi.org/10.21203/rs.3.pex-2607/v1), to the new version (Version 3) of the article that will be submitted shortly. That being said, once the concentrated cell media is collected, the protein concentration is determined using Bradford reagent protein assay, and the concentrations are adjusted to ensure equal protein amounts are loaded into the gel. We understand the authors were not clear on this aspect of the sample preparation procedure for western blot and have included this to the Methods section in the new version of the article.

Reviewer Comment:
Furthermore, the description of Figure 1 B refers to cell lysates:
" Lysates of HAP1 (WT and MDK KO) were prepared, and ~30 μg of protein was processed for Western blot."
This is not explained in the experimental section. This should definitely be explained or corrected.

Author Response:
To the “Antibody screening by Western blot on culture media” area of the methods section, we have clarified that 30 μg of protein was added to each lane of the polyacrylamide gel. We’ve also referenced our full detailed procedure, which includes troubleshooting when unprecedented results or issues arise.
Competing Interests: No competing interests were disclosed. Close
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Author Response 01 Aug 2024

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

01 Aug 2024

Author Response

The authors would like to clarify our response to Jens-Ulrich Rahfeld in regards to cell density and amount of protein used.

HAP1 cells (WT and MDK KO) were grown in ... Continue reading The authors would like to clarify our response to Jens-Ulrich Rahfeld in regards to cell density and amount of protein used.

HAP1 cells (WT and MDK KO) were grown in 20 ml complete medium to 80% confluence in 150 mm dishes. At 80% confluency, HAP1 cells were at a density of ~1.0 x 10⁶/ml (~20.0 x 10⁶/ 20ml). To prepare for protein extraction, cells were washed 3x with PBS and starved for ~18 hrs. Culture media were collected and centrifuged for 10 min at 500 x g to eliminate cells and larger contaminants, then for 10 min at 4500 x g to eliminate smaller contaminants. Culture media were initially concentrated using Amicon Ultra-15 Centrifugal Filter Units (MilliporeSigma cat. number UFC9010) by centrifuging at 4000 x g for 15min. The resulting 500 μl of concentrated media from each filter unit were centrifuged again at 4000 x g for 15 min using Amicon Ultra- 0.5 Centrifugal Filter Units (MilliporeSigma cat. number UFC5010) to 200 μl. The final protein concentration was 0.3 mg/ml and 100 μl (30 μg) of media were used for each blot.

An updated version of the manuscript has been submitted and we have included this protocol for cell preparation in the Methods section.
The authors would like to clarify our response to Jens-Ulrich Rahfeld in regards to cell density and amount of protein used.

HAP1 cells (WT and MDK KO) were grown in 20 ml complete medium to 80% confluence in 150 mm dishes. At 80% confluency, HAP1 cells were at a density of ~1.0 x 10⁶/ml (~20.0 x 10⁶/ 20ml). To prepare for protein extraction, cells were washed 3x with PBS and starved for ~18 hrs. Culture media were collected and centrifuged for 10 min at 500 x g to eliminate cells and larger contaminants, then for 10 min at 4500 x g to eliminate smaller contaminants. Culture media were initially concentrated using Amicon Ultra-15 Centrifugal Filter Units (MilliporeSigma cat. number UFC9010) by centrifuging at 4000 x g for 15min. The resulting 500 μl of concentrated media from each filter unit were centrifuged again at 4000 x g for 15 min using Amicon Ultra- 0.5 Centrifugal Filter Units (MilliporeSigma cat. number UFC5010) to 200 μl. The final protein concentration was 0.3 mg/ml and 100 μl (30 μg) of media were used for each blot.

An updated version of the manuscript has been submitted and we have included this protocol for cell preparation in the Methods section.
Competing Interests: No competing interests were disclosed. Close
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Author Response 23 Jul 2024

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

23 Jul 2024

Author Response

Thank you to Dr. Jens-Ulrich Rahfeld for your comprehensive review of this article which presents as a guide to selecting high-performing antibodies for Midkine to progress research of this target ... Continue reading Thank you to Dr. Jens-Ulrich Rahfeld for your comprehensive review of this article which presents as a guide to selecting high-performing antibodies for Midkine to progress research of this target associated to many pathological conditions. We hope our response to your comments (italicized) successfully answers any concerns you previously had.

Reviewer Comment:
However, there are ambiguities in the experimental and methodological part:
The protocol mentions that the cell culture supernatants were obtained from starving cultures and then concentrated (also shown in scheme A in Figure 1.
For the reproducibility of the preparation of these supernatants, there is no information on the cell density/ml of the cultivated HAP1 cells or the initial volume of the cell culture supernatant concentrated for analysis.

Author Response:
To facilitate reproducibility of the antibody characterization platform utilized by the authors, we have included a reference to the full procedure, available on Protocol exchange (https://doi.org/10.21203/rs.3.pex-2607/v1), to the new version (Version 3) of the article that will be submitted shortly. That being said, once the concentrated cell media is collected, the protein concentration is determined using Bradford reagent protein assay, and the concentrations are adjusted to ensure equal protein amounts are loaded into the gel. We understand the authors were not clear on this aspect of the sample preparation procedure for western blot and have included this to the Methods section in the new version of the article.

Reviewer Comment:
Furthermore, the description of Figure 1 B refers to cell lysates:
" Lysates of HAP1 (WT and MDK KO) were prepared, and ~30 μg of protein was processed for Western blot."
This is not explained in the experimental section. This should definitely be explained or corrected.

Author Response:
To the “Antibody screening by Western blot on culture media” area of the methods section, we have clarified that 30 μg of protein was added to each lane of the polyacrylamide gel. We’ve also referenced our full detailed procedure, which includes troubleshooting when unprecedented results or issues arise.
Thank you to Dr. Jens-Ulrich Rahfeld for your comprehensive review of this article which presents as a guide to selecting high-performing antibodies for Midkine to progress research of this target associated to many pathological conditions. We hope our response to your comments (italicized) successfully answers any concerns you previously had.

Reviewer Comment:
However, there are ambiguities in the experimental and methodological part:
The protocol mentions that the cell culture supernatants were obtained from starving cultures and then concentrated (also shown in scheme A in Figure 1.
For the reproducibility of the preparation of these supernatants, there is no information on the cell density/ml of the cultivated HAP1 cells or the initial volume of the cell culture supernatant concentrated for analysis.

Author Response:
To facilitate reproducibility of the antibody characterization platform utilized by the authors, we have included a reference to the full procedure, available on Protocol exchange (https://doi.org/10.21203/rs.3.pex-2607/v1), to the new version (Version 3) of the article that will be submitted shortly. That being said, once the concentrated cell media is collected, the protein concentration is determined using Bradford reagent protein assay, and the concentrations are adjusted to ensure equal protein amounts are loaded into the gel. We understand the authors were not clear on this aspect of the sample preparation procedure for western blot and have included this to the Methods section in the new version of the article.

Reviewer Comment:
Furthermore, the description of Figure 1 B refers to cell lysates:
" Lysates of HAP1 (WT and MDK KO) were prepared, and ~30 μg of protein was processed for Western blot."
This is not explained in the experimental section. This should definitely be explained or corrected.

Author Response:
To the “Antibody screening by Western blot on culture media” area of the methods section, we have clarified that 30 μg of protein was added to each lane of the polyacrylamide gel. We’ve also referenced our full detailed procedure, which includes troubleshooting when unprecedented results or issues arise.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Author Response 01 Aug 2024

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

01 Aug 2024

Author Response

The authors would like to clarify our response to Jens-Ulrich Rahfeld in regards to cell density and amount of protein used.

HAP1 cells (WT and MDK KO) were grown in ... Continue reading The authors would like to clarify our response to Jens-Ulrich Rahfeld in regards to cell density and amount of protein used.

HAP1 cells (WT and MDK KO) were grown in 20 ml complete medium to 80% confluence in 150 mm dishes. At 80% confluency, HAP1 cells were at a density of ~1.0 x 10⁶/ml (~20.0 x 10⁶/ 20ml). To prepare for protein extraction, cells were washed 3x with PBS and starved for ~18 hrs. Culture media were collected and centrifuged for 10 min at 500 x g to eliminate cells and larger contaminants, then for 10 min at 4500 x g to eliminate smaller contaminants. Culture media were initially concentrated using Amicon Ultra-15 Centrifugal Filter Units (MilliporeSigma cat. number UFC9010) by centrifuging at 4000 x g for 15min. The resulting 500 μl of concentrated media from each filter unit were centrifuged again at 4000 x g for 15 min using Amicon Ultra- 0.5 Centrifugal Filter Units (MilliporeSigma cat. number UFC5010) to 200 μl. The final protein concentration was 0.3 mg/ml and 100 μl (30 μg) of media were used for each blot.

An updated version of the manuscript has been submitted and we have included this protocol for cell preparation in the Methods section.
The authors would like to clarify our response to Jens-Ulrich Rahfeld in regards to cell density and amount of protein used.

HAP1 cells (WT and MDK KO) were grown in 20 ml complete medium to 80% confluence in 150 mm dishes. At 80% confluency, HAP1 cells were at a density of ~1.0 x 10⁶/ml (~20.0 x 10⁶/ 20ml). To prepare for protein extraction, cells were washed 3x with PBS and starved for ~18 hrs. Culture media were collected and centrifuged for 10 min at 500 x g to eliminate cells and larger contaminants, then for 10 min at 4500 x g to eliminate smaller contaminants. Culture media were initially concentrated using Amicon Ultra-15 Centrifugal Filter Units (MilliporeSigma cat. number UFC9010) by centrifuging at 4000 x g for 15min. The resulting 500 μl of concentrated media from each filter unit were centrifuged again at 4000 x g for 15 min using Amicon Ultra- 0.5 Centrifugal Filter Units (MilliporeSigma cat. number UFC5010) to 200 μl. The final protein concentration was 0.3 mg/ml and 100 μl (30 μg) of media were used for each blot.

An updated version of the manuscript has been submitted and we have included this protocol for cell preparation in the Methods section.
Competing Interests: No competing interests were disclosed. Close
Report a concern

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Reviewer Report 18 Jan 2024

Jean-Pierre Bellier, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA

Approved

https://doi.org/10.5256/f1000research.160165.r231070

I have ... Continue reading

CITE

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VERSION 1

PUBLISHED 09 Feb 2023

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Reviewer Report 29 Nov 2023

Jean-Pierre Bellier, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.143357.r208220

In this manuscript, Ayoubi et al. investigated the specificity of eight commercially available antibodies directed against the human Midkine protein. Midkine is a secreted pleiotropic protein involved in cell proliferation, survival, and the regulation of inflammation. It is often associated with tissue repair and cancer development and has also been shown to be present in amyloid-beta plaques in Alzheimer's disease.

The manuscript is well-written, with experiments meticulously executed and technically sound, utilizing HAP1 cell WT and KO as controls. The availability of all datasets on Zenodo is highly commendable and professional.

This work reveals that among the antibodies tested, three performed well for Western blotting (WB), and while all showed positive signals for immunoprecipitation (IP), six completely depleted the supernatant of the culture from Midkine protein. Two antibodies performed well in both experiments. Although the authors do not provide a detailed commentary and interpretation of their results, the data presentation is clear enough for readers to identify potentially suitable antibodies for WB and IP studies.

Specific comments:

It would have been valuable if the authors had conducted a similar comparison for Immunofluorescence using WT and KO HAP1 cells.
For greater clarity, the authors should specify in the title that their study pertains to human Midkine and mention that HAP1 is a human cell line.
The term "high performing" used in the title may be misleading since the study primarily assesses antibody specificity rather than overall performance. If the authors want to use “High-performance” in the title, they should consider providing some interpretation of their data in the discussion.
In the introduction, the authors mentioned that Midkine (MK) is a potential biomarker and therapeutic target for Alzheimer's disease (AD). The original reference for the work demonstrating MK in senile plaques is "Yasuhara, O., Muramatsu, H., Kim, S. U., Muramatsu, T., Maruta, H., and McGeer, P. L. (1993) Midkine, a novel neurotrophic factor, is present in senile plaques of Alzheimer's disease. Biochem. Biophys. Res. Commun. 192, 246-251. [PMID: 8476427]"
Two of the antibodies featured in this study, NBP2-66948 and GTX116089, are discontinued. The authors should mention this.
Have the authors optimized the working concentration used? If so, they should explain the optimization process.
The authors should also provide a rationale for selecting the antibodies tested. Many of them are not commonly cited in the literature. An explanation for their choice of antibodies would enhance the manuscript's comprehensibility.
Finally, could the authors clarify why it is necessary to deplete the cells? Is it to promote Midkine secretion or to avoid interference from IgG in subsequent experiments? Further clarification on this point would be beneficial.
Some references (10-13) are not complete.

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

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

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

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

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Neuroscience, antibody generation and use.

CITE

Report a concern

Author Response 18 Jan 2024

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

18 Jan 2024

Author Response

Dear Jean-Pierre Bellier,

Thank you for your feedback. We have submitted a modified version of this manuscript, with your requested modifications included. We trust that the refinements made meet ... Continue reading Dear Jean-Pierre Bellier,

Thank you for your feedback. We have submitted a modified version of this manuscript, with your requested modifications included. We trust that the refinements made meet your expectations, enhance comprehensibility and address any concerns you might have had. Please see our responses to your specific comments below.

1. It would have been valuable if the authors had conducted a similar comparison for Immunofluorescence using WT and KO HAP1 cells.
We haven’t been able to detect intracellular Midkine by Western Blot. This is because once Midkine is synthesized into a protein, it is quickly secreted. Due to this factor, we expect that detecting intracellular Midkine via Immunofluorescence would be very difficult given the fixation and permeabilization methods used with our standard protocol.

2. For greater clarity the authors should specify in the title that their study pertains to human midkine and mention HAP1 is a human cell line.
In the newly submitted version of this article, the title has been updated to indicate that our study pertains to human Midkine. In the results and discussion section, we have clarified that the HAP1 cell line used is human. Thank you for this suggestion.

3.The term "high performing" used in the title may be misleading since the study primarily assesses antibody specificity rather than overall performance. If the authors want to use “High-performance” in the title, they should consider providing some interpretation of their data in the discussion.
The new title of this article, “A guide to selecting high-performing antibodies for human Midkine for use in Western Blot and immunoprecipitation”. This modification aims to convey that the article’s objective is to guide researchers in selecting high-performing antibodies for their research’s needs. To the introduction, we have included a statement which indicates why the authors chose not to assess the results and draw conclusions on the antibodies performance. We trust these adjustments meet your expectations and prevent any further misinterpretations.

4.In the introduction, the authors mentioned that Midkine (MK) is a potential biomarker and therapeutic target for Alzheimer's disease (AD). The original reference for the work demonstrating MK in senile plaques is "Yasuhara, O., Muramatsu, H., Kim, S. U., Muramatsu, T., Maruta, H., and McGeer, P. L. (1993) Midkine, a novel neurotrophic factor, is present in senile plaques of Alzheimer's disease. Biochem. Biophys. Res. Commun. 192, 246-251. [PMID: 8476427]"
Thank you for providing the original reference for the work done to isolate MDK in senile plaques. It has been included in the new version as reference number 7.

5.Two of the antibodies featured in this study, NBP2-66948 and GTX116089, are discontinued. The authors should mention this.
This piece of information has been included in Table 2, where discontinued antibodies are now indicated with a superscript a.

6.Have the authors optimized the working concentration used? If so, they should explain the optimization process.
This study for Midkine is part of a much larger collaborative initiative, seeking to characterize antibodies for all human proteins to address the antibody liability crisis. It would be very difficult to optimize every working parameter when in the process of trying to characterize antibodies for 20,000 proteins in the human proteome. That being said, we have developed universal protocols that has been successful for characterizing antibodies for 85 proteins thus far. In our experiments, a major parameter that we can control to optimize the process is the level of endogeneous protein in the selected cell type. We try to select cell lines, using DepMap portal, with adequate transcript levels of the protein target.

7.The authors should also provide a rationale for selecting the antibodies tested. Many of them are not commonly cited in the literature. An explanation for their choice of antibodies would enhance the manuscript's comprehensibility.
The antibodies to be characterization under our standard procedure are donated by YCharOS partners (https://ycharos.com/partners/). These industry partners cover nearly 28% of all antibodies listed on the Antibody registry (antibodyregistry.org). When the industry partners are informed of upcoming targets, they donate the antibodies they have the capacity to supply at the time the study commences.

8.Finally, could the authors clarify why it is necessary to deplete the cells? Is it to promote Midkine secretion or to avoid interference from IgG in subsequent experiments? Further clarification on this point would be beneficial.
To help us provide a complete response to this comment, could you specify what you mean by “deplete the cells”?

9.Some references (10-13) are not complete.
The DOI for citations pertaining to our prior work, available on Zenodo, have been included in the new version submitted. While these references are not found on PubMed, and may seem “incomplete”, they are crucial in helping readers comprehend the protocols used and access the underlying data.
Dear Jean-Pierre Bellier,

Thank you for your feedback. We have submitted a modified version of this manuscript, with your requested modifications included. We trust that the refinements made meet your expectations, enhance comprehensibility and address any concerns you might have had. Please see our responses to your specific comments below.

1. It would have been valuable if the authors had conducted a similar comparison for Immunofluorescence using WT and KO HAP1 cells.
We haven’t been able to detect intracellular Midkine by Western Blot. This is because once Midkine is synthesized into a protein, it is quickly secreted. Due to this factor, we expect that detecting intracellular Midkine via Immunofluorescence would be very difficult given the fixation and permeabilization methods used with our standard protocol.

2. For greater clarity the authors should specify in the title that their study pertains to human midkine and mention HAP1 is a human cell line.
In the newly submitted version of this article, the title has been updated to indicate that our study pertains to human Midkine. In the results and discussion section, we have clarified that the HAP1 cell line used is human. Thank you for this suggestion.

3.The term "high performing" used in the title may be misleading since the study primarily assesses antibody specificity rather than overall performance. If the authors want to use “High-performance” in the title, they should consider providing some interpretation of their data in the discussion.
The new title of this article, “A guide to selecting high-performing antibodies for human Midkine for use in Western Blot and immunoprecipitation”. This modification aims to convey that the article’s objective is to guide researchers in selecting high-performing antibodies for their research’s needs. To the introduction, we have included a statement which indicates why the authors chose not to assess the results and draw conclusions on the antibodies performance. We trust these adjustments meet your expectations and prevent any further misinterpretations.

4.In the introduction, the authors mentioned that Midkine (MK) is a potential biomarker and therapeutic target for Alzheimer's disease (AD). The original reference for the work demonstrating MK in senile plaques is "Yasuhara, O., Muramatsu, H., Kim, S. U., Muramatsu, T., Maruta, H., and McGeer, P. L. (1993) Midkine, a novel neurotrophic factor, is present in senile plaques of Alzheimer's disease. Biochem. Biophys. Res. Commun. 192, 246-251. [PMID: 8476427]"
Thank you for providing the original reference for the work done to isolate MDK in senile plaques. It has been included in the new version as reference number 7.

5.Two of the antibodies featured in this study, NBP2-66948 and GTX116089, are discontinued. The authors should mention this.
This piece of information has been included in Table 2, where discontinued antibodies are now indicated with a superscript a.

6.Have the authors optimized the working concentration used? If so, they should explain the optimization process.
This study for Midkine is part of a much larger collaborative initiative, seeking to characterize antibodies for all human proteins to address the antibody liability crisis. It would be very difficult to optimize every working parameter when in the process of trying to characterize antibodies for 20,000 proteins in the human proteome. That being said, we have developed universal protocols that has been successful for characterizing antibodies for 85 proteins thus far. In our experiments, a major parameter that we can control to optimize the process is the level of endogeneous protein in the selected cell type. We try to select cell lines, using DepMap portal, with adequate transcript levels of the protein target.

7.The authors should also provide a rationale for selecting the antibodies tested. Many of them are not commonly cited in the literature. An explanation for their choice of antibodies would enhance the manuscript's comprehensibility.
The antibodies to be characterization under our standard procedure are donated by YCharOS partners (https://ycharos.com/partners/). These industry partners cover nearly 28% of all antibodies listed on the Antibody registry (antibodyregistry.org). When the industry partners are informed of upcoming targets, they donate the antibodies they have the capacity to supply at the time the study commences.

8.Finally, could the authors clarify why it is necessary to deplete the cells? Is it to promote Midkine secretion or to avoid interference from IgG in subsequent experiments? Further clarification on this point would be beneficial.
To help us provide a complete response to this comment, could you specify what you mean by “deplete the cells”?

9.Some references (10-13) are not complete.
The DOI for citations pertaining to our prior work, available on Zenodo, have been included in the new version submitted. While these references are not found on PubMed, and may seem “incomplete”, they are crucial in helping readers comprehend the protocols used and access the underlying data.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 18 Jan 2024

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

18 Jan 2024

Author Response

Dear Jean-Pierre Bellier,

Thank you for your feedback. We have submitted a modified version of this manuscript, with your requested modifications included. We trust that the refinements made meet ... Continue reading Dear Jean-Pierre Bellier,

Thank you for your feedback. We have submitted a modified version of this manuscript, with your requested modifications included. We trust that the refinements made meet your expectations, enhance comprehensibility and address any concerns you might have had. Please see our responses to your specific comments below.

1. It would have been valuable if the authors had conducted a similar comparison for Immunofluorescence using WT and KO HAP1 cells.
We haven’t been able to detect intracellular Midkine by Western Blot. This is because once Midkine is synthesized into a protein, it is quickly secreted. Due to this factor, we expect that detecting intracellular Midkine via Immunofluorescence would be very difficult given the fixation and permeabilization methods used with our standard protocol.

2. For greater clarity the authors should specify in the title that their study pertains to human midkine and mention HAP1 is a human cell line.
In the newly submitted version of this article, the title has been updated to indicate that our study pertains to human Midkine. In the results and discussion section, we have clarified that the HAP1 cell line used is human. Thank you for this suggestion.

3.The term "high performing" used in the title may be misleading since the study primarily assesses antibody specificity rather than overall performance. If the authors want to use “High-performance” in the title, they should consider providing some interpretation of their data in the discussion.
The new title of this article, “A guide to selecting high-performing antibodies for human Midkine for use in Western Blot and immunoprecipitation”. This modification aims to convey that the article’s objective is to guide researchers in selecting high-performing antibodies for their research’s needs. To the introduction, we have included a statement which indicates why the authors chose not to assess the results and draw conclusions on the antibodies performance. We trust these adjustments meet your expectations and prevent any further misinterpretations.

4.In the introduction, the authors mentioned that Midkine (MK) is a potential biomarker and therapeutic target for Alzheimer's disease (AD). The original reference for the work demonstrating MK in senile plaques is "Yasuhara, O., Muramatsu, H., Kim, S. U., Muramatsu, T., Maruta, H., and McGeer, P. L. (1993) Midkine, a novel neurotrophic factor, is present in senile plaques of Alzheimer's disease. Biochem. Biophys. Res. Commun. 192, 246-251. [PMID: 8476427]"
Thank you for providing the original reference for the work done to isolate MDK in senile plaques. It has been included in the new version as reference number 7.

5.Two of the antibodies featured in this study, NBP2-66948 and GTX116089, are discontinued. The authors should mention this.
This piece of information has been included in Table 2, where discontinued antibodies are now indicated with a superscript a.

6.Have the authors optimized the working concentration used? If so, they should explain the optimization process.
This study for Midkine is part of a much larger collaborative initiative, seeking to characterize antibodies for all human proteins to address the antibody liability crisis. It would be very difficult to optimize every working parameter when in the process of trying to characterize antibodies for 20,000 proteins in the human proteome. That being said, we have developed universal protocols that has been successful for characterizing antibodies for 85 proteins thus far. In our experiments, a major parameter that we can control to optimize the process is the level of endogeneous protein in the selected cell type. We try to select cell lines, using DepMap portal, with adequate transcript levels of the protein target.

7.The authors should also provide a rationale for selecting the antibodies tested. Many of them are not commonly cited in the literature. An explanation for their choice of antibodies would enhance the manuscript's comprehensibility.
The antibodies to be characterization under our standard procedure are donated by YCharOS partners (https://ycharos.com/partners/). These industry partners cover nearly 28% of all antibodies listed on the Antibody registry (antibodyregistry.org). When the industry partners are informed of upcoming targets, they donate the antibodies they have the capacity to supply at the time the study commences.

8.Finally, could the authors clarify why it is necessary to deplete the cells? Is it to promote Midkine secretion or to avoid interference from IgG in subsequent experiments? Further clarification on this point would be beneficial.
To help us provide a complete response to this comment, could you specify what you mean by “deplete the cells”?

9.Some references (10-13) are not complete.
The DOI for citations pertaining to our prior work, available on Zenodo, have been included in the new version submitted. While these references are not found on PubMed, and may seem “incomplete”, they are crucial in helping readers comprehend the protocols used and access the underlying data.
Dear Jean-Pierre Bellier,

Thank you for your feedback. We have submitted a modified version of this manuscript, with your requested modifications included. We trust that the refinements made meet your expectations, enhance comprehensibility and address any concerns you might have had. Please see our responses to your specific comments below.

1. It would have been valuable if the authors had conducted a similar comparison for Immunofluorescence using WT and KO HAP1 cells.
We haven’t been able to detect intracellular Midkine by Western Blot. This is because once Midkine is synthesized into a protein, it is quickly secreted. Due to this factor, we expect that detecting intracellular Midkine via Immunofluorescence would be very difficult given the fixation and permeabilization methods used with our standard protocol.

2. For greater clarity the authors should specify in the title that their study pertains to human midkine and mention HAP1 is a human cell line.
In the newly submitted version of this article, the title has been updated to indicate that our study pertains to human Midkine. In the results and discussion section, we have clarified that the HAP1 cell line used is human. Thank you for this suggestion.

3.The term "high performing" used in the title may be misleading since the study primarily assesses antibody specificity rather than overall performance. If the authors want to use “High-performance” in the title, they should consider providing some interpretation of their data in the discussion.
The new title of this article, “A guide to selecting high-performing antibodies for human Midkine for use in Western Blot and immunoprecipitation”. This modification aims to convey that the article’s objective is to guide researchers in selecting high-performing antibodies for their research’s needs. To the introduction, we have included a statement which indicates why the authors chose not to assess the results and draw conclusions on the antibodies performance. We trust these adjustments meet your expectations and prevent any further misinterpretations.

4.In the introduction, the authors mentioned that Midkine (MK) is a potential biomarker and therapeutic target for Alzheimer's disease (AD). The original reference for the work demonstrating MK in senile plaques is "Yasuhara, O., Muramatsu, H., Kim, S. U., Muramatsu, T., Maruta, H., and McGeer, P. L. (1993) Midkine, a novel neurotrophic factor, is present in senile plaques of Alzheimer's disease. Biochem. Biophys. Res. Commun. 192, 246-251. [PMID: 8476427]"
Thank you for providing the original reference for the work done to isolate MDK in senile plaques. It has been included in the new version as reference number 7.

5.Two of the antibodies featured in this study, NBP2-66948 and GTX116089, are discontinued. The authors should mention this.
This piece of information has been included in Table 2, where discontinued antibodies are now indicated with a superscript a.

6.Have the authors optimized the working concentration used? If so, they should explain the optimization process.
This study for Midkine is part of a much larger collaborative initiative, seeking to characterize antibodies for all human proteins to address the antibody liability crisis. It would be very difficult to optimize every working parameter when in the process of trying to characterize antibodies for 20,000 proteins in the human proteome. That being said, we have developed universal protocols that has been successful for characterizing antibodies for 85 proteins thus far. In our experiments, a major parameter that we can control to optimize the process is the level of endogeneous protein in the selected cell type. We try to select cell lines, using DepMap portal, with adequate transcript levels of the protein target.

7.The authors should also provide a rationale for selecting the antibodies tested. Many of them are not commonly cited in the literature. An explanation for their choice of antibodies would enhance the manuscript's comprehensibility.
The antibodies to be characterization under our standard procedure are donated by YCharOS partners (https://ycharos.com/partners/). These industry partners cover nearly 28% of all antibodies listed on the Antibody registry (antibodyregistry.org). When the industry partners are informed of upcoming targets, they donate the antibodies they have the capacity to supply at the time the study commences.

8.Finally, could the authors clarify why it is necessary to deplete the cells? Is it to promote Midkine secretion or to avoid interference from IgG in subsequent experiments? Further clarification on this point would be beneficial.
To help us provide a complete response to this comment, could you specify what you mean by “deplete the cells”?

9.Some references (10-13) are not complete.
The DOI for citations pertaining to our prior work, available on Zenodo, have been included in the new version submitted. While these references are not found on PubMed, and may seem “incomplete”, they are crucial in helping readers comprehend the protocols used and access the underlying data.
Competing Interests: No competing interests were disclosed. Close
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VERSION 4 PUBLISHED 09 Feb 2023

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Jean-Pierre Bellier, Brigham and Women's Hospital, Boston, MA, USA
Jens-Ulrich Rahfeld, Fraunhofer Institute for Cell Therapy and Immunology, Department Molecular Drug Biochemistry and Therapy, Weinbergweg 22, 06120, Halle, Germany

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31 Jul 2024 | for Version 3

Jens-Ulrich Rahfeld, Fraunhofer Institute for Cell Therapy and Immunology, Department Molecular Drug Biochemistry and Therapy, Weinbergweg 22, 06120, Halle, Germany

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Approved

Okay, no further comments!
Greetings
Jens Rahfeld

Competing Interests

No competing interests were disclosed.

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proteinchemisty

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13 May 2024 | for Version 2

Jens-Ulrich Rahfeld, Fraunhofer Institute for Cell Therapy and Immunology, Department Molecular Drug Biochemistry and Therapy, Weinbergweg 22, 06120, Halle, Germany

20 Views Cite this report Responses(2)

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?

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

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

proteinchemisty

Respond to this report

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Author Response

23 Jul 2024

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

Thank you to Dr. Jens-Ulrich Rahfeld for your comprehensive review of this article which presents as a guide to selecting high-performing antibodies for Midkine to progress research of this target associated to many pathological conditions. We hope our response to your comments (italicized) successfully answers any concerns you previously had.

Reviewer Comment:
However, there are ambiguities in the experimental and methodological part:
The protocol mentions that the cell culture supernatants were obtained from starving cultures and then concentrated (also shown in scheme A in Figure 1.
For the reproducibility of the preparation of these supernatants, there is no information on the cell density/ml of the cultivated HAP1 cells or the initial volume of the cell culture supernatant concentrated for analysis.

Author Response:
To facilitate reproducibility of the antibody characterization platform utilized by the authors, we have included a reference to the full procedure, available on Protocol exchange (https://doi.org/10.21203/rs.3.pex-2607/v1), to the new version (Version 3) of the article that will be submitted shortly. That being said, once the concentrated cell media is collected, the protein concentration is determined using Bradford reagent protein assay, and the concentrations are adjusted to ensure equal protein amounts are loaded into the gel. We understand the authors were not clear on this aspect of the sample preparation procedure for western blot and have included this to the Methods section in the new version of the article.

Reviewer Comment:
Furthermore, the description of Figure 1 B refers to cell lysates:
" Lysates of HAP1 (WT and MDK KO) were prepared, and ~30 μg of protein was processed for Western blot."
This is not explained in the experimental section. This should definitely be explained or corrected.

Author Response:
To the “Antibody screening by Western blot on culture media” area of the methods section, we have clarified that 30 μg of protein was added to each lane of the polyacrylamide gel. We’ve also referenced our full detailed procedure, which includes troubleshooting when unprecedented results or issues arise.

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Competing Interests

No competing interests were disclosed.

Author Response

01 Aug 2024

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

The authors would like to clarify our response to Jens-Ulrich Rahfeld in regards to cell density and amount of protein used.

HAP1 cells (WT and MDK KO) were grown in 20 ml complete medium to 80% confluence in 150 mm dishes. At 80% confluency, HAP1 cells were at a density of ~1.0 x 10⁶/ml (~20.0 x 10⁶/ 20ml). To prepare for protein extraction, cells were washed 3x with PBS and starved for ~18 hrs. Culture media were collected and centrifuged for 10 min at 500 x g to eliminate cells and larger contaminants, then for 10 min at 4500 x g to eliminate smaller contaminants. Culture media were initially concentrated using Amicon Ultra-15 Centrifugal Filter Units (MilliporeSigma cat. number UFC9010) by centrifuging at 4000 x g for 15min. The resulting 500 μl of concentrated media from each filter unit were centrifuged again at 4000 x g for 15 min using Amicon Ultra- 0.5 Centrifugal Filter Units (MilliporeSigma cat. number UFC5010) to 200 μl. The final protein concentration was 0.3 mg/ml and 100 μl (30 μg) of media were used for each blot.

An updated version of the manuscript has been submitted and we have included this protocol for cell preparation in the Methods section.

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Competing Interests

No competing interests were disclosed.

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Reviewer Report

8 Views

18 Jan 2024 | for Version 2

Jean-Pierre Bellier, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA

8 Views Cite this report Responses(0)

Approved

I have no further comments.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Neuroscience, antibody generation and use.

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

21 Views

29 Nov 2023 | for Version 1

Jean-Pierre Bellier, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA

21 Views Cite this report Responses(1)

Approved With Reservations

It would have been valuable if the authors had conducted a similar comparison for Immunofluorescence using WT and KO HAP1 cells.
For greater clarity, the authors should specify in the title that their study pertains to human Midkine and mention that HAP1 is a human cell line.
The term "high performing" used in the title may be misleading since the study primarily assesses antibody specificity rather than overall performance. If the authors want to use “High-performance” in the title, they should consider providing some interpretation of their data in the discussion.
In the introduction, the authors mentioned that Midkine (MK) is a potential biomarker and therapeutic target for Alzheimer's disease (AD). The original reference for the work demonstrating MK in senile plaques is "Yasuhara, O., Muramatsu, H., Kim, S. U., Muramatsu, T., Maruta, H., and McGeer, P. L. (1993) Midkine, a novel neurotrophic factor, is present in senile plaques of Alzheimer's disease. Biochem. Biophys. Res. Commun. 192, 246-251. [PMID: 8476427]"
Two of the antibodies featured in this study, NBP2-66948 and GTX116089, are discontinued. The authors should mention this.
Have the authors optimized the working concentration used? If so, they should explain the optimization process.
The authors should also provide a rationale for selecting the antibodies tested. Many of them are not commonly cited in the literature. An explanation for their choice of antibodies would enhance the manuscript's comprehensibility.
Finally, could the authors clarify why it is necessary to deplete the cells? Is it to promote Midkine secretion or to avoid interference from IgG in subsequent experiments? Further clarification on this point would be beneficial.
Some references (10-13) are not complete.

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

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

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

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

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Neuroscience, antibody generation and use.

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Responses (1)

Author Response

18 Jan 2024

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

Dear Jean-Pierre Bellier,

Thank you for your feedback. We have submitted a modified version of this manuscript, with your requested modifications included. We trust that the refinements made meet your expectations, enhance comprehensibility and address any concerns you might have had. Please see our responses to your specific comments below.

1. It would have been valuable if the authors had conducted a similar comparison for Immunofluorescence using WT and KO HAP1 cells.
We haven’t been able to detect intracellular Midkine by Western Blot. This is because once Midkine is synthesized into a protein, it is quickly secreted. Due to this factor, we expect that detecting intracellular Midkine via Immunofluorescence would be very difficult given the fixation and permeabilization methods used with our standard protocol.

2. For greater clarity the authors should specify in the title that their study pertains to human midkine and mention HAP1 is a human cell line.
In the newly submitted version of this article, the title has been updated to indicate that our study pertains to human Midkine. In the results and discussion section, we have clarified that the HAP1 cell line used is human. Thank you for this suggestion.

3.The term "high performing" used in the title may be misleading since the study primarily assesses antibody specificity rather than overall performance. If the authors want to use “High-performance” in the title, they should consider providing some interpretation of their data in the discussion.
The new title of this article, “A guide to selecting high-performing antibodies for human Midkine for use in Western Blot and immunoprecipitation”. This modification aims to convey that the article’s objective is to guide researchers in selecting high-performing antibodies for their research’s needs. To the introduction, we have included a statement which indicates why the authors chose not to assess the results and draw conclusions on the antibodies performance. We trust these adjustments meet your expectations and prevent any further misinterpretations.

4.In the introduction, the authors mentioned that Midkine (MK) is a potential biomarker and therapeutic target for Alzheimer's disease (AD). The original reference for the work demonstrating MK in senile plaques is "Yasuhara, O., Muramatsu, H., Kim, S. U., Muramatsu, T., Maruta, H., and McGeer, P. L. (1993) Midkine, a novel neurotrophic factor, is present in senile plaques of Alzheimer's disease. Biochem. Biophys. Res. Commun. 192, 246-251. [PMID: 8476427]"
Thank you for providing the original reference for the work done to isolate MDK in senile plaques. It has been included in the new version as reference number 7.

5.Two of the antibodies featured in this study, NBP2-66948 and GTX116089, are discontinued. The authors should mention this.
This piece of information has been included in Table 2, where discontinued antibodies are now indicated with a superscript a.

6.Have the authors optimized the working concentration used? If so, they should explain the optimization process.
This study for Midkine is part of a much larger collaborative initiative, seeking to characterize antibodies for all human proteins to address the antibody liability crisis. It would be very difficult to optimize every working parameter when in the process of trying to characterize antibodies for 20,000 proteins in the human proteome. That being said, we have developed universal protocols that has been successful for characterizing antibodies for 85 proteins thus far. In our experiments, a major parameter that we can control to optimize the process is the level of endogeneous protein in the selected cell type. We try to select cell lines, using DepMap portal, with adequate transcript levels of the protein target.

7.The authors should also provide a rationale for selecting the antibodies tested. Many of them are not commonly cited in the literature. An explanation for their choice of antibodies would enhance the manuscript's comprehensibility.
The antibodies to be characterization under our standard procedure are donated by YCharOS partners (https://ycharos.com/partners/). These industry partners cover nearly 28% of all antibodies listed on the Antibody registry (antibodyregistry.org). When the industry partners are informed of upcoming targets, they donate the antibodies they have the capacity to supply at the time the study commences.

8.Finally, could the authors clarify why it is necessary to deplete the cells? Is it to promote Midkine secretion or to avoid interference from IgG in subsequent experiments? Further clarification on this point would be beneficial.
To help us provide a complete response to this comment, could you specify what you mean by “deplete the cells”?

9.Some references (10-13) are not complete.
The DOI for citations pertaining to our prior work, available on Zenodo, have been included in the new version submitted. While these references are not found on PubMed, and may seem “incomplete”, they are crucial in helping readers comprehend the protocols used and access the underlying data.

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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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A guide to selecting high-performing antibodies for human Midkine for use in Western blot and immunoprecipitation

Abstract

Keywords

Revised Amendments from Version 3

Introduction

Results and discussion

Table 1. Summary of the cell lines used.

Table 2. Summary of the Midkine antibodies tested.

Figure 1. Midkine antibody screening by Western blot on culture media.

Figure 2. Midkine antibody screening by immunoprecipitation on culture media.

Methods

Antibodies

Cell culture

Antibody screening by Western blot on culture medium

Antibody screening by immunoprecipitation on culture medium

Data availability

Underlying data

Acknowledgment

References

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