A guide to selecting high-performing antibodies for CSNK2A1 (UniProt ID: P68400) for use in western blot, immunoprecipitation and immunofluorescence

Riham Ayoubi; Maryam Fotouhi; Charles Alende; Vera Ruíz Moleón; Kathleen Southern; Carl Laflamme; NeuroSGC/YCharOS/EDDU collaborative group; ABIF consortium

doi:10.12688/f1000research.153243.1

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Data Note

A guide to selecting high-performing antibodies for CSNK2A1 (UniProt ID: P68400) for use in western blot, immunoprecipitation and immunofluorescence

[version 1; peer review: 3 approved with reservations]

Riham Ayoubi¹, Maryam Fotouhi¹, Charles Alende¹, [...] Vera Ruíz Moleón¹, Kathleen Southern¹, Carl Laflamme ¹, NeuroSGC/YCharOS/EDDU collaborative group, ABIF consortium

Riham Ayoubi¹, Maryam Fotouhi¹, [...] Charles Alende¹, Vera Ruíz Moleón¹, Kathleen Southern¹, Carl Laflamme ¹, NeuroSGC/YCharOS/EDDU collaborative group, ABIF consortium

PUBLISHED 09 Jul 2024

Author details Author details

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

Riham Ayoubi
Roles: Investigation, Methodology, Validation, Visualization

Maryam Fotouhi
Roles: Investigation

Charles Alende
Roles: Investigation, Visualization

Vera Ruíz Moleón
Roles: Investigation

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

Carl Laflamme
Roles: Formal Analysis, Funding Acquisition, Project Administration, Resources, Supervision, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

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

Abstract

Casein kinase II subunit alpha (CSNK2A1), a serine/threonine kinase, phosphorylates multiple protein substrates and is involved in diverse cellular and biological processes. Implicated in various human diseases, high-performing antibodies would help evaluate its potential as a therapeutic target and benefit the scientific community. In this study, we have characterized ten CSNK2A1 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Keywords

UniProt ID P68400, CSNK2A1, Casein kinase II subunit alpha, antibody characterization, antibody validation, western blot, immunoprecipitation, immunofluorescence

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 -Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex -Horizon Discovery -Proteintech -Synaptic Systems -Thermo Fisher Scientific.

Grant information: This study was funded in part by the Simons Foundation Autism Research Initiative (SFARI). It was also partly funded by a grant from Canadian Institutes of Health Research Foundation (grant no. FDN154305) and by the Government of Canada through Genome Canada, Genome Quebec and Ontario Genomics (grant no. OGI-210). RA is supported by a Mitacs fellowship. 
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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, Fotouhi M, Alende C et al. A guide to selecting high-performing antibodies for CSNK2A1 (UniProt ID: P68400) for use in western blot, immunoprecipitation and immunofluorescence [version 1; peer review: 3 approved with reservations]. F1000Research 2024, 13:781 (https://doi.org/10.12688/f1000research.153243.1) First published: 09 Jul 2024, 13:781 (https://doi.org/10.12688/f1000research.153243.1) Latest published: 05 Sep 2024, 13:781 (https://doi.org/10.12688/f1000research.153243.2)

Introduction

Casein kinase II subunit alpha (CSNK2A1), encoded by the CSNK2A1 gene, is a catalytic subunit of the serine/threonine enzyme, casein kinase 2; essential for cell cycle progression, apoptosis, transcription and viral replication.¹^–⁵ Relevant to the etiology of many diseases, including the identification of two missense mutations in the CSNK2A1 gene associated with autism spectrum disorder, CSNK2A1 is emerging as a promising biomarker and therapeutic target.¹^,⁶^–¹⁷ High-performing antibodies would enable data reproducibility and reliable research findings.

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.¹⁸^–²⁰ Here, we evaluated the performance of ten commercially-available antibodies for CSNK2A1 for use in western blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of the proteins properties and function. The platform for antibody characterization used to carry out this study was endorsed by a committee of industry and academic representatives. It consists of identifying human cell lines with adequate target protein expression and the development/contribution of equivalent knockout (KO) cell lines, followed by antibody characterization procedures with most of the commercially available antibodies against the corresponding target protein. The standardized consensus antibody characterization protocols are openly available on Protocol Exchange (DOI: 10.21203/rs.3.pex-2607/v1).²¹

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. Guidelines on how to interpret antibody characterization data found in this study are featured on the YCharOS gateway.²²

Results and discussion

Our standard protocol involves comparing readouts from wild-type (WT) and KO cells.²³^,²⁴ The first step is to identify a cell line(s) that expresses sufficient levels of CSNK2A1 to generate a measurable signal using antibodies. 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). The HAP1 cell lines expresses the CSNK2A1 transcript at 7.0 log₂ (TPM+1) RNA levels, which is above the average range of cancer cells analyzed. Parental and CSNK2A KO 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	HZGHC004051c003	CVCL_SJ92	HAP1	CSNK2A1

For western blot experiments, WT and CSNK2A KO protein lysates were ran on SDS-PAGE, transferrred onto nitrocellulose membranes, and then probed with ten CSNK2A1 antibodies in parallel (Table 2, Figure 1).

Table 2. Summary of the CSNK2A1 antibodies tested.

Company	Catalog number	Lot number	RRID (Antibody Registry)	Clonality	Clone ID	Host	Concentration (μg/μl)	Vendors recommended applications
Abcam	ab76040**	1001668-2	AB_1523361	recombinant-mono	EP1963Y	rabbit	0.16	Wb
Abcam	ab236664	1012742-3	AB_3073947	polyclonal	-	rabbit	2.0	Wb, IP, IF
Bio-Techne	MAB7957*	CHSN0121081	AB_3073948	monoclonal	844720	mouse	0.5	Wb
Bio-Techne	NBP3-19853**	230458	AB_3073949	recombinant-mono	S05-7F8	rabbit	0.3	Wb
Cell Signaling Technology	2656	3	AB_2236816	polyclonal	-	rabbit	0.03	Wb
Genetex	GTX107576	40366	AB_10616991	polyclonal	-	rabbit	1.0	Wb
Genetex	GTX107897	40002	AB_1950048	polyclonal	-	rabbit	0.62	Wb, IF
Genetex	GTX107949	39869	AB_2036686	polyclonal	-	rabbit	0.2	Wb
Proteintech	68200-1-Ig*	10028709	AB_2935289	monoclonal	1D5E8	mouse	1.0	Wb
Thermo Fisher Scientific	702811**	2062784	AB_2734801	recombinant-mono	7H29L3	rabbit	0.5	Wb

* Monoclonal antibody.

** Recombinant antibody.

Figure 1. CSNK2A1 antibody screening by western blot.

Lysates of HAP1 (WT and CSNK2A KO) were prepared and 30 μg of protein were processed for western blot with the indicated CSNK2A1 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 polyacrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. An exception was given to 68200-1-Ig* recommended at 1/20 000, as the signal was too weak and was therefore diluted and was used at 1/10 000. Antibody dilution used: ab76040** at 1/500, ab236664 at 1/1000, MAB7957* at 1/1000, NBP3-19853** at 1/1000, 2656 at 1/500, GTX107576 at 1/500, GTX107897 at 1/500, GTX107949 at 1/500, 68200-1-Ig* at 1/10 000, 702811** at 1/10 000. Predicted band size: 45 kDa. *Monoclonal antibody, **Recombinant antibody.

We then assessed the capability of all ten antibodies to capture CSNK2A1 from HAP1 protein extracts using immunoprecipitation techniques, followed by western blot analysis. For the immunoblot step, a specific CSNK2A1 antibody identified previously (refer to Figure 1) was selected. Equal amounts of the starting material (SM), the unbound fraction (UB), as well as the whole immunoprecipitate (IP) eluates were separated by SDS-PAGE (Figure 2).

Figure 2. CSNK2A1 antibody screening by immunoprecipitation.

HAP1 lysates were prepared, and immunoprecipitation was performed using 2.0 μg of the indicated CSNK2A1 antibodies pre-coupled to Dynabeads protein G or protein A. Samples were washed and processed for western blot with the indicated CSNK2A1 antibody. For western blot, 702811** was used at 1/10 000. The ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM = 4% starting material; UB = 4% unbound fraction; IP = immunoprecipitate, HC = antibody heavy chain. *Monoclonal antibody, **Recombinant antibody.

For immunofluorescence, ten antibodies were screened using a mosaic strategy. First, HAP1 WT and CSNK2A1 KO cells were labelled with distinct fluorescent dyes in order to distinguish the two cell lines, and the ten CSNK2A1 antibodies were evaluated. Both WT and KO lines were imaged in the same field of view to reduce staining, imaging and image analysis bias (Figure 3). Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested.²¹ The images presented in Figure 3 are representative of the results of this analysis.

Figure 3. CSNK2A1 antibody screening by immunofluorescence.

HAP1 WT and CSNK2A1 KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio in a 96-well plate with an optically clear flat-bottom. Cells were stained with the indicated CSNK2A1 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (identification of WT cells), red (antibody staining) and far-red (identification of KO cells) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, respectively. When the concentration was not indicated by the supplier, antibodies were tested at concentrations where the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: ab76040** at 1/500, ab236664 at 1/100, MAB7957* at 1/500, NBP3-19853** at 1/300, 2656 at 1/30, GTX107576 at 1/100, GTX107897 at 1/100, GTX107949 at 1/200, 68200-1-Ig* at 1/500, 702811** at 1/250. Bars = 10 μm. *Monoclonal antibody, **Recombinant antibody.

In conclusion, we have screened ten CSNK2A1 commercial antibodies by western blot, immunoprecipitation and immunofluorescence. Several high-quality antibodies that successfully detect CSNK2A1 under our standardized experimental conditions can be identified. Researchers who wish to study CSNK2A1 in a different species are encouraged to select high-quality antibodies, based on the results of this study, and investigate the predicted species reactivity of the manufacturer before extending their research.

The underlying data for this study can be found on Zenodo, an open-access repository for which YCharOS has its own collection of antibody characterization reports.²⁷^,²⁸

Methods

The standardized protocols used to carry out this KO cell line-based antibody characterization platform was established and approved by a collaborative group of academics, industry researchers and antibody manufacturers. The detailed materials and step-by-step protocols used to characterize antibodies in western blot, immunoprecipitation and immunofluorescence are openly available on Protocol Exchange, a repository dedicated to openly sharing scientific research protocols (DOI: 10.21203/rs.3.pex-2607/v1).²¹

Antibodies and cell lines used

Cell lines used and primary antibodies tested in this study are listed in Table 1 and 2, respectively. To ensure that the cell lines and antibodies are cited properly and can be easily identified, we have included their corresponding Research Resource Identifiers, or RRID.²⁵^,²⁶

Data availability

Underlying data

Zenodo: Antibody Characterization Report for CSNK2A1, doi.org/10.5281/zenodo.10818214.²⁷

Zenodo: Dataset for the CSNK2A1 antibody screening study, doi.org/10.5281/zenodo.11078556.²⁸

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

Acknowledgment

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

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

ABIF consortium: Claire M. Brown and Joel Ryan.

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

An earlier version of this of this article can be found on Zenodo (doi: 10.5281/zenodo.10818214).

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Version 2

VERSION 2 PUBLISHED 09 Jul 2024

Author details Author details

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

Riham Ayoubi
Roles: Investigation, Methodology, Validation, Visualization

Maryam Fotouhi
Roles: Investigation

Charles Alende
Roles: Investigation, Visualization

Vera Ruíz Moleón
Roles: Investigation

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

Carl Laflamme
Roles: Formal Analysis, Funding Acquisition, Project Administration, Resources, Supervision, 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 -Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex -Horizon Discovery -Proteintech -Synaptic Systems -Thermo Fisher Scientific.

Grant information

This study was funded in part by the Simons Foundation Autism Research Initiative (SFARI). It was also partly funded by a grant from Canadian Institutes of Health Research Foundation (grant no. FDN154305) and by the Government of Canada through Genome Canada, Genome Quebec and Ontario Genomics (grant no. OGI-210). RA is supported by a Mitacs fellowship. 
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (2)

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Published: 05 Sep 2024, 13:781

https://doi.org/10.12688/f1000research.153243.2

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Published: 09 Jul 2024, 13:781

https://doi.org/10.12688/f1000research.153243.1

© 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, Fotouhi M, Alende C et al. A guide to selecting high-performing antibodies for CSNK2A1 (UniProt ID: P68400) for use in western blot, immunoprecipitation and immunofluorescence [version 1; peer review: 3 approved with reservations]. F1000Research 2024, 13:781 (https://doi.org/10.12688/f1000research.153243.1)

NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.

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Version 1

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Reviewer Report 27 Aug 2024

Miwako Homma, Fukushima Medical University School of Medicine, Fukushima, Japan

Approved with Reservations

https://doi.org/10.5256/f1000research.168109.r303889

This is an interesting study in an area that needs investigating. CK2(CSNK2A1) protein is the first enzyme found to have phosphorylation activity in eukaryotic cells, and it has been shown to participate in various major biological processes. Since there have been several reports of pathologies caused by CSNK2A1 gene mutations, such as Okur-Chung neurodevelopmental syndrome, autism and cancer, high-quality, high-performance antibodies against CSNK2A1 protein are expected to advance research on these diseases. Therefore, the authors conducted the following studies. They characterized ten commercially available antibodies against CSNK2A1 for use: 1. in western blotting under conditions that detect denatured intracellular proteins, 2. in immunoprecipitation to demonstrate antibody recognition of native CSNK2A1 in vivo, and 3. in immunofluorescence for cell imaging. However, a few points require clarification and further validation.

Antibody evaluations discussed in this manuscript are only comparative observations, and titers of individual antibodies are not quantified. As shown in Figure 1, only one concentration of each antibody diluent was selected, according to the supplier's recommendations. Affinity binding properties are generally verified quantitatively by ELISA, along with Western blotting, in which the intensity of each antibody to the target protein, in this case, purified CSNK2A1, can be calculated by stepwise dilution. I recommend that the authors compare each antibody at the concentration that maximizes performance and that blots be further quantified. It is interesting to note that there are few commercial antibodies that can immunoprecipitate the target molecule, CSNK2A1. However, immunoprecipitation was performed using only one concentration of the indicated antibodies.
Although CSNK2A1 (CK2) and CSNK2A2 (CK2’) are encoded at different loci, the amino acid sequences are very similar. Therefore, it is necessary to show that these antibodies are specific for CSNK2A1(CK2) and that they do not also recognize CSNK2A2 (CK2’). For this purpose, I recommend using cells with CSNK2A1 genetically knocked-out (ko) as a control, to show that antibody responses are not observed when only endogenous CANK2A2 is present, or when CSNK2A2 is expressed as a tagged cDNA in ko cells. Alternatively, antibody performance should be validated using human CSNK2A1 or CSNK2A2 recombinant proteins.

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: Intracellular signal transduction, protein kinases, CK2, phospho-proteomics, CK2-ChIP-Seq, RNA-Seq, epigenetics, and prognostic biomarker of cancer.

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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Reviewer Report 02 Aug 2024

David Litchfield, Western University, London, Ontario, Canada

Approved with Reservations

https://doi.org/10.5256/f1000research.168109.r303880

This Data Note presents a systematic characterization of a number of commercially available antibodies directed against the gene product of CSNK2A1 (also referred to as the alpha subunit of Casein Kinase II). As noted by the authors, CSNK2A1 is involved in numerous fundamental biological processes and has been implicated as a promising therapeutic target. High-quality antibodies against CSNK2A1 and reliable protocols for the use of these antibodies are therefore of considerable importance both for elucidating its biological functions and for advancing efforts to explore the promise of CSNK2A1 as a therapeutic target. Utilization of knockout cell lines to demonstrate that the antibody signal is dependent on the expression of CSNK2A1 provides particularly compelling validation of the specificity of the antibodies. To further enhance the utility of this report and its accessibility to the research community, I can offer the following suggestions.

Considering that the gene product encoded by CSNK2A1 is referred to by a variety of names in the published literature in addition to terms adopted by UniProt, it may be beneficial to include additional terms (including CK2alpha and/or CK2α as well as CKIIalpha or CKIIα that are commonly used in the literature). This suggestion is intended to maximize visibility of this article using different search engines.
The CSNK2A1 and CSNK2A2 genes encode proteins with very similar enzymatic characteristics and a very high degree of sequence similarity especially within their catalytic kinase domains. The main differences between the gene products encoded by CSNK2A1 and CSNK2A2 reside within their distinct C-terminal domains that extend beyond their kinase domains. In reviewing the technical information for some of the antibodies that was available on vendor websites, which in some cases was very limited due to proprietary considerations, it was not entirely clear to me whether some of these antibodies (particularly polyclonal antibodies and/or antibodies raised against recombinant proteins encompassing regions of very high similarity between CSNK2A1 and CSNK2A2) would also detect CSNK2A2 (ie. CK2α’). Overall, although it does not appear that CSNK2A2 (which represents a protein that is 41 amino acids shorter than the protein encoded by CSNK2A1) is detected by any of the antibodies in Western blots, I believe it would be beneficial to make direct reference to CSNK2A2 in this article and highlight this issue as a potential limitation and/or issue that warrants consideration. This issue would be particularly pertinent for studies to be performed in the absence of the paired cell lines (i.e., parental and knockout) as used in this report.

Minor suggestions.

Introduction (line 1) – need to add “kinase” following “serine/threonine” (either to replace “enzyme” or in addition to including “enzyme”)
Introduction (line 2). I would recommend replacing “essential” with less definitive terminology (potentially “with essential roles in”) since CSNK2A1 may not be universally involved in all of the listed processes.
Results and Discussion; Last sentence of 1^st paragraph and 1^st line of 2^nd paragraph; should be CSNK2A1 (instead of CSNK2A) – especially considering the existence of CSNK2A2 as noted above.
Results and Discussion. 2^rd paragraph. “were separated on SDS-PAGE” instead of “ran on SDS-PAGE”
Results and Discussion. 3^rd paragraph. “equal proportions” rather than “equal amounts”.

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: Areas of expertise include signal transduction, protein kinases, kinase inhibitors and phosphoproteomics. The CK2 family of protein kinases (including CSNK2A1 and CSNK2A2) have represented a central focus of our research.

CITE

Report a concern

Author Response 05 Sep 2024

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

05 Sep 2024

Author Response
Thank you to David Litchfield for reviewing this manuscript. We hope our response to your comments and concerns clarify any misinterpretations and questions you may have previously had. Additionally, the ... Continue reading
Thank you to David Litchfield for reviewing this manuscript. We hope our response to your comments and concerns clarify any misinterpretations and questions you may have previously had. Additionally, the authors have revisited the initial manuscript, including a detailed methods section to ensure the procedure is reproducible. As well, a limitation section has been included to address concerns regarding the inherent limitations of the overall platform as well as when evaluating CSNK2A1 antibodies. A third table has been included to help guide readers interpret the results and select high-performing antibodies, which is the aim of this study. Please refer to version 2.

Considering that the gene product encoded by CSNK2A1 is referred to by a variety of names in the published literature in addition to terms adopted by UniProt, it may be beneficial to include additional terms (including CK2alpha and/or CK2α as well as CKIIalpha or CKIIα that are commonly used in the literature). This suggestion is intended to maximize visibility of this article using different search engines.

Thank you for this suggestion, we are always looking to broaden our audience to increase end-user accessibility of our characterization data to enable researchers to select high-quality antibodies for their research. To this end, we have included the suggested terms to our list of keywords. Please refer to version 2.

2. The CSNK2A1 and CSNK2A2 genes encode proteins with very similar enzymatic characteristics and a very high degree of sequence similarity especially within their catalytic kinase domains. The main differences between the gene products encoded by CSNK2A1 and CSNK2A2 reside within their distinct C-terminal domains that extend beyond their kinase domains. In reviewing the technical information for some of the antibodies that was available on vendor websites, which in some cases was very limited due to proprietary considerations, it was not entirely clear to me whether some of these antibodies (particularly polyclonal antibodies and/or antibodies raised against recombinant proteins encompassing regions of very high similarity between CSNK2A1 and CSNK2A2) would also detect CSNK2A2 (ie. CK2α’). Overall, although it does not appear that CSNK2A2 (which represents a protein that is 41 amino acids shorter than the protein encoded by CSNK2A1) is detected by any of the antibodies in Western blots, I believe it would be beneficial to make direct reference to CSNK2A2 in this article and highlight this issue as a potential limitation and/or issue that warrants consideration. This issue would be particularly pertinent for studies to be performed in the absence of the paired cell lines (i.e., parental and knockout) as used in this report.

The authors are aware of the potential cross reactivity of the antibodies with CSNK2A2. The advantage of using genetic strategies (WT vs KO) for characterization is that we are able to detect whether the antibodies can or cannot target CSNK2A1 by assessing whether the signal is lost in the KO line. That being said, transparency and reproducibility is at the core of our initiative’s values which is why we have mentioned this factor in the limitations section, included in version 2 of the manuscript.

Minor suggestions.

Introduction (line 1) – need to add “kinase” following “serine/threonine” (either to replace “enzyme” or in addition to including “enzyme”)

Introduction (line 2). I would recommend replacing “essential” with less definitive terminology (potentially “with essential roles in”) since CSNK2A1 may not be universally involved in all of the listed processes.

Results and Discussion; Last sentence of 1^st paragraph and 1^stline of 2^nd paragraph; should be CSNK2A1 (instead of CSNK2A) – especially considering the existence of CSNK2A2 as noted above.

Results and Discussion. 2^rdparagraph. “were separated on SDS-PAGE” instead of “ran on SDS-PAGE”

Results and Discussion. 3^rdparagraph. “equal proportions” rather than “equal amounts”.

All of these minor changes have been attended to in version 2 of the manuscript which has been submitted.
Thank you to David Litchfield for reviewing this manuscript. We hope our response to your comments and concerns clarify any misinterpretations and questions you may have previously had. Additionally, the authors have revisited the initial manuscript, including a detailed methods section to ensure the procedure is reproducible. As well, a limitation section has been included to address concerns regarding the inherent limitations of the overall platform as well as when evaluating CSNK2A1 antibodies. A third table has been included to help guide readers interpret the results and select high-performing antibodies, which is the aim of this study. Please refer to version 2.

Considering that the gene product encoded by CSNK2A1 is referred to by a variety of names in the published literature in addition to terms adopted by UniProt, it may be beneficial to include additional terms (including CK2alpha and/or CK2α as well as CKIIalpha or CKIIα that are commonly used in the literature). This suggestion is intended to maximize visibility of this article using different search engines.

Thank you for this suggestion, we are always looking to broaden our audience to increase end-user accessibility of our characterization data to enable researchers to select high-quality antibodies for their research. To this end, we have included the suggested terms to our list of keywords. Please refer to version 2.

2. The CSNK2A1 and CSNK2A2 genes encode proteins with very similar enzymatic characteristics and a very high degree of sequence similarity especially within their catalytic kinase domains. The main differences between the gene products encoded by CSNK2A1 and CSNK2A2 reside within their distinct C-terminal domains that extend beyond their kinase domains. In reviewing the technical information for some of the antibodies that was available on vendor websites, which in some cases was very limited due to proprietary considerations, it was not entirely clear to me whether some of these antibodies (particularly polyclonal antibodies and/or antibodies raised against recombinant proteins encompassing regions of very high similarity between CSNK2A1 and CSNK2A2) would also detect CSNK2A2 (ie. CK2α’). Overall, although it does not appear that CSNK2A2 (which represents a protein that is 41 amino acids shorter than the protein encoded by CSNK2A1) is detected by any of the antibodies in Western blots, I believe it would be beneficial to make direct reference to CSNK2A2 in this article and highlight this issue as a potential limitation and/or issue that warrants consideration. This issue would be particularly pertinent for studies to be performed in the absence of the paired cell lines (i.e., parental and knockout) as used in this report.

The authors are aware of the potential cross reactivity of the antibodies with CSNK2A2. The advantage of using genetic strategies (WT vs KO) for characterization is that we are able to detect whether the antibodies can or cannot target CSNK2A1 by assessing whether the signal is lost in the KO line. That being said, transparency and reproducibility is at the core of our initiative’s values which is why we have mentioned this factor in the limitations section, included in version 2 of the manuscript.

Minor suggestions.

Introduction (line 1) – need to add “kinase” following “serine/threonine” (either to replace “enzyme” or in addition to including “enzyme”)

Introduction (line 2). I would recommend replacing “essential” with less definitive terminology (potentially “with essential roles in”) since CSNK2A1 may not be universally involved in all of the listed processes.

Results and Discussion; Last sentence of 1^st paragraph and 1^stline of 2^nd paragraph; should be CSNK2A1 (instead of CSNK2A) – especially considering the existence of CSNK2A2 as noted above.

Results and Discussion. 2^rdparagraph. “were separated on SDS-PAGE” instead of “ran on SDS-PAGE”

Results and Discussion. 3^rdparagraph. “equal proportions” rather than “equal amounts”.

All of these minor changes have been attended to in version 2 of the manuscript which has been submitted.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 05 Sep 2024

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

05 Sep 2024

Author Response
Thank you to David Litchfield for reviewing this manuscript. We hope our response to your comments and concerns clarify any misinterpretations and questions you may have previously had. Additionally, the ... Continue reading
Thank you to David Litchfield for reviewing this manuscript. We hope our response to your comments and concerns clarify any misinterpretations and questions you may have previously had. Additionally, the authors have revisited the initial manuscript, including a detailed methods section to ensure the procedure is reproducible. As well, a limitation section has been included to address concerns regarding the inherent limitations of the overall platform as well as when evaluating CSNK2A1 antibodies. A third table has been included to help guide readers interpret the results and select high-performing antibodies, which is the aim of this study. Please refer to version 2.

Considering that the gene product encoded by CSNK2A1 is referred to by a variety of names in the published literature in addition to terms adopted by UniProt, it may be beneficial to include additional terms (including CK2alpha and/or CK2α as well as CKIIalpha or CKIIα that are commonly used in the literature). This suggestion is intended to maximize visibility of this article using different search engines.

Thank you for this suggestion, we are always looking to broaden our audience to increase end-user accessibility of our characterization data to enable researchers to select high-quality antibodies for their research. To this end, we have included the suggested terms to our list of keywords. Please refer to version 2.

2. The CSNK2A1 and CSNK2A2 genes encode proteins with very similar enzymatic characteristics and a very high degree of sequence similarity especially within their catalytic kinase domains. The main differences between the gene products encoded by CSNK2A1 and CSNK2A2 reside within their distinct C-terminal domains that extend beyond their kinase domains. In reviewing the technical information for some of the antibodies that was available on vendor websites, which in some cases was very limited due to proprietary considerations, it was not entirely clear to me whether some of these antibodies (particularly polyclonal antibodies and/or antibodies raised against recombinant proteins encompassing regions of very high similarity between CSNK2A1 and CSNK2A2) would also detect CSNK2A2 (ie. CK2α’). Overall, although it does not appear that CSNK2A2 (which represents a protein that is 41 amino acids shorter than the protein encoded by CSNK2A1) is detected by any of the antibodies in Western blots, I believe it would be beneficial to make direct reference to CSNK2A2 in this article and highlight this issue as a potential limitation and/or issue that warrants consideration. This issue would be particularly pertinent for studies to be performed in the absence of the paired cell lines (i.e., parental and knockout) as used in this report.

The authors are aware of the potential cross reactivity of the antibodies with CSNK2A2. The advantage of using genetic strategies (WT vs KO) for characterization is that we are able to detect whether the antibodies can or cannot target CSNK2A1 by assessing whether the signal is lost in the KO line. That being said, transparency and reproducibility is at the core of our initiative’s values which is why we have mentioned this factor in the limitations section, included in version 2 of the manuscript.

Minor suggestions.

Introduction (line 1) – need to add “kinase” following “serine/threonine” (either to replace “enzyme” or in addition to including “enzyme”)

Introduction (line 2). I would recommend replacing “essential” with less definitive terminology (potentially “with essential roles in”) since CSNK2A1 may not be universally involved in all of the listed processes.

Results and Discussion; Last sentence of 1^st paragraph and 1^stline of 2^nd paragraph; should be CSNK2A1 (instead of CSNK2A) – especially considering the existence of CSNK2A2 as noted above.

Results and Discussion. 2^rdparagraph. “were separated on SDS-PAGE” instead of “ran on SDS-PAGE”

Results and Discussion. 3^rdparagraph. “equal proportions” rather than “equal amounts”.

All of these minor changes have been attended to in version 2 of the manuscript which has been submitted.
Thank you to David Litchfield for reviewing this manuscript. We hope our response to your comments and concerns clarify any misinterpretations and questions you may have previously had. Additionally, the authors have revisited the initial manuscript, including a detailed methods section to ensure the procedure is reproducible. As well, a limitation section has been included to address concerns regarding the inherent limitations of the overall platform as well as when evaluating CSNK2A1 antibodies. A third table has been included to help guide readers interpret the results and select high-performing antibodies, which is the aim of this study. Please refer to version 2.

Considering that the gene product encoded by CSNK2A1 is referred to by a variety of names in the published literature in addition to terms adopted by UniProt, it may be beneficial to include additional terms (including CK2alpha and/or CK2α as well as CKIIalpha or CKIIα that are commonly used in the literature). This suggestion is intended to maximize visibility of this article using different search engines.

Thank you for this suggestion, we are always looking to broaden our audience to increase end-user accessibility of our characterization data to enable researchers to select high-quality antibodies for their research. To this end, we have included the suggested terms to our list of keywords. Please refer to version 2.

2. The CSNK2A1 and CSNK2A2 genes encode proteins with very similar enzymatic characteristics and a very high degree of sequence similarity especially within their catalytic kinase domains. The main differences between the gene products encoded by CSNK2A1 and CSNK2A2 reside within their distinct C-terminal domains that extend beyond their kinase domains. In reviewing the technical information for some of the antibodies that was available on vendor websites, which in some cases was very limited due to proprietary considerations, it was not entirely clear to me whether some of these antibodies (particularly polyclonal antibodies and/or antibodies raised against recombinant proteins encompassing regions of very high similarity between CSNK2A1 and CSNK2A2) would also detect CSNK2A2 (ie. CK2α’). Overall, although it does not appear that CSNK2A2 (which represents a protein that is 41 amino acids shorter than the protein encoded by CSNK2A1) is detected by any of the antibodies in Western blots, I believe it would be beneficial to make direct reference to CSNK2A2 in this article and highlight this issue as a potential limitation and/or issue that warrants consideration. This issue would be particularly pertinent for studies to be performed in the absence of the paired cell lines (i.e., parental and knockout) as used in this report.

The authors are aware of the potential cross reactivity of the antibodies with CSNK2A2. The advantage of using genetic strategies (WT vs KO) for characterization is that we are able to detect whether the antibodies can or cannot target CSNK2A1 by assessing whether the signal is lost in the KO line. That being said, transparency and reproducibility is at the core of our initiative’s values which is why we have mentioned this factor in the limitations section, included in version 2 of the manuscript.

Minor suggestions.

Introduction (line 1) – need to add “kinase” following “serine/threonine” (either to replace “enzyme” or in addition to including “enzyme”)

Introduction (line 2). I would recommend replacing “essential” with less definitive terminology (potentially “with essential roles in”) since CSNK2A1 may not be universally involved in all of the listed processes.

Results and Discussion; Last sentence of 1^st paragraph and 1^stline of 2^nd paragraph; should be CSNK2A1 (instead of CSNK2A) – especially considering the existence of CSNK2A2 as noted above.

Results and Discussion. 2^rdparagraph. “were separated on SDS-PAGE” instead of “ran on SDS-PAGE”

Results and Discussion. 3^rdparagraph. “equal proportions” rather than “equal amounts”.

All of these minor changes have been attended to in version 2 of the manuscript which has been submitted.
Competing Interests: No competing interests were disclosed. Close
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Reviewer Report 01 Aug 2024

Odile Filhol, Université Grenoble-Alpes, Grenoble-Alpes, France

Catherine Pillet, French Alternative Energies and Atomic Energy Commission, Grenoble, France

Approved with Reservations

https://doi.org/10.5256/f1000research.168109.r303886

The article untitled “A guide to selecting high-performing antibodies for CSNK2A1 for use in western blot, immunoprecipitation and immunofluorescence” describes the characterization of ten CSNK2A1 commercially available antibodies and their indicated protocols.
As written in the abstract, these studies are part of a larger collaborative initiative that is very interesting, and useful, especially in the case of Casein Kinase 2 for which, some antibodies are sometime not adapted.
The major concern for me, as a specialist of CK2, is that there is not at least, a comment on the specificity of the antibodies to recognize the CSNK2A2 isoform, that is highly homolog to CSNK2A1. Does the HAP1 cell line used by the authors, express this isoform?
In the immunoprecipitation assays illustrated in Figure 2, 2 µg of each antibody was used. How did you decide to select 2 µg of antibody to coupled them on beads? However, either in Ponceau red staining or using the same antibody for western blot detection, for some of them, the heavy chain antibody is not visible. This is probably due to the low binding of the antibody. Could you please comment on that in the manuscript?
In Figure 3, could you show a least one picture representative of the HAP1 WT (green) and CSNK2A1 KO (far red) cells that allow identifying them.
Minor points:
In table 2, stars in the second column are not useful since the information is provided in column 5. Stars and the corresponding legend should be removed.
In Figure 1 and 3, the antibody dilution should be indicated together with the name of each antibody.
Except the points raised in the comments above, the results are well presented.
However, I would have liked to have a useful conclusion, suggesting the best CK2 antibodies in relation to their different applications.

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: Cell biology and protein kinase CK2 specialist.

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.

CITE

Report a concern

Author Response 05 Sep 2024

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

05 Sep 2024

Author Response

Thank you to Odile Filhol and Catherine Pillet for your thorough analysis of our manuscript presenting antibody characterization data for ten commercial CSNK2A1 antibodies to the scientific community. We hope ... Continue reading Thank you to Odile Filhol and Catherine Pillet for your thorough analysis of our manuscript presenting antibody characterization data for ten commercial CSNK2A1 antibodies to the scientific community. We hope our response to your comments and concerns clarify any misinterpretations and questions you may have previously had. Additionally, the authors have revisited the initial manuscript and made necessary changes to include a detailed methods section as to ensure the procedure is reproducible. As well, a limitation section has been included to address concerns regarding the nature of the platform and inherent limitations associated with it. Please refer to version 2.

Major points:
The major concern for me, as a specialist of CK2, is that there is not at least, a comment on the specificity of the antibodies to recognize the CSNK2A2 isoform, that is highly homolog to CSNK2A1. Does the HAP1 cell line used by the authors, express this isoform?

Author Response: Based on the characterization table provided by Depmap, HAP1 expresses the CSNK2A2 isoform at 5.8 log₂(TPM+1). That being said, this study is shared with the scientific community to help researchers find selective antibodies, already commercially available, for CSNK2A1. That is why a CSNK2A1 KO cell line was selected. To test the ability of the antibodies to recognize the isoform, we would need to test them in a CSNK2A2 KO line.

In the immunoprecipitation assays illustrated in Figure 2, 2 µg of each antibody was used. How did you decide to select 2 µg of antibody to coupled them on beads? However, either in Ponceau red staining or using the same antibody for western blot detection, for some of them, the heavy chain antibody is not visible. This is probably due to the low binding of the antibody. Could you please comment on that in the manuscript?

Author Response: The amount of 2 µg is selected based on previous experience with trial and error. 1ug was tested in the past and optimized to 2 µg to limit cross reactivity in the IP lane when performing the western blot for IP.

As for the ponceau staining, the initial concentrations provided by the manufacturers are followed when adding the 2 µg of antibody. In some cases, adhering to the manufacturers antibody concentrations can result in concentrations that are highly diluted. For example, the 2656 antibody from Cell Signalling Technology sis provided at a concentration of 0.03 µg/ul. Given this low concentration, the amount of antibody is very minimal preventing the heavy chain from being visible.

In Figure 3, could you show a least one picture representative of the HAP1 WT (green) and CSNK2A1 KO (far red) cells that allow identifying them.

Author Response: All of the underlying data is available in the Dataset, referenced in this manuscript and available on the YCharOS zenodo community. Please refer to the following link: https://doi.org/10.5281/zenodo.11078556 . The addition of Table 3 should also clarify how the WT and KO cells can be identified.

Minor points:
In table 2, stars in the second column are not useful since the information is provided in column 5. Stars and the corresponding legend should be removed.

Author Response: The stars are used outside throughout the report to highlight the renewable antibodies while also allowing the readers to remember the clonality of each antibody without having to refer back to Table 2.

In Figure 1 and 3, the antibody dilution should be indicated together with the name of each antibody.

Author Response: The antibody dilutions are indicated with the catalog number of each antibody in the figure legends.

Except the points raised in the comments above, the results are well presented.
However, I would have liked to have a useful conclusion, suggesting the best CK2 antibodies in relation to their different applications.

Author Response: We understand this concern and it is why we have chosen to submit the antibody characterization data in the format of Data Notes. As explained in the F1000 article guidelines, Data Notes do not include result analyses or conclusions. Furthermore, the authors prefer to remain unbiased while being transparent with the results obtained from the experiments. To provide context for readers who do not have experience analyzing results from such characterization methods utilizing knockdown lines as controls, we have included a subsequent Table to demonstrate how one can identify successful vs unsuccessful antibodies in each application. Please refer to Table 3 in the updated submission.
Thank you to Odile Filhol and Catherine Pillet for your thorough analysis of our manuscript presenting antibody characterization data for ten commercial CSNK2A1 antibodies to the scientific community. We hope our response to your comments and concerns clarify any misinterpretations and questions you may have previously had. Additionally, the authors have revisited the initial manuscript and made necessary changes to include a detailed methods section as to ensure the procedure is reproducible. As well, a limitation section has been included to address concerns regarding the nature of the platform and inherent limitations associated with it. Please refer to version 2.

Major points:
The major concern for me, as a specialist of CK2, is that there is not at least, a comment on the specificity of the antibodies to recognize the CSNK2A2 isoform, that is highly homolog to CSNK2A1. Does the HAP1 cell line used by the authors, express this isoform?

Author Response: Based on the characterization table provided by Depmap, HAP1 expresses the CSNK2A2 isoform at 5.8 log₂(TPM+1). That being said, this study is shared with the scientific community to help researchers find selective antibodies, already commercially available, for CSNK2A1. That is why a CSNK2A1 KO cell line was selected. To test the ability of the antibodies to recognize the isoform, we would need to test them in a CSNK2A2 KO line.

In the immunoprecipitation assays illustrated in Figure 2, 2 µg of each antibody was used. How did you decide to select 2 µg of antibody to coupled them on beads? However, either in Ponceau red staining or using the same antibody for western blot detection, for some of them, the heavy chain antibody is not visible. This is probably due to the low binding of the antibody. Could you please comment on that in the manuscript?

Author Response: The amount of 2 µg is selected based on previous experience with trial and error. 1ug was tested in the past and optimized to 2 µg to limit cross reactivity in the IP lane when performing the western blot for IP.

As for the ponceau staining, the initial concentrations provided by the manufacturers are followed when adding the 2 µg of antibody. In some cases, adhering to the manufacturers antibody concentrations can result in concentrations that are highly diluted. For example, the 2656 antibody from Cell Signalling Technology sis provided at a concentration of 0.03 µg/ul. Given this low concentration, the amount of antibody is very minimal preventing the heavy chain from being visible.

In Figure 3, could you show a least one picture representative of the HAP1 WT (green) and CSNK2A1 KO (far red) cells that allow identifying them.

Author Response: All of the underlying data is available in the Dataset, referenced in this manuscript and available on the YCharOS zenodo community. Please refer to the following link: https://doi.org/10.5281/zenodo.11078556 . The addition of Table 3 should also clarify how the WT and KO cells can be identified.

Minor points:
In table 2, stars in the second column are not useful since the information is provided in column 5. Stars and the corresponding legend should be removed.

Author Response: The stars are used outside throughout the report to highlight the renewable antibodies while also allowing the readers to remember the clonality of each antibody without having to refer back to Table 2.

In Figure 1 and 3, the antibody dilution should be indicated together with the name of each antibody.

Author Response: The antibody dilutions are indicated with the catalog number of each antibody in the figure legends.

Except the points raised in the comments above, the results are well presented.
However, I would have liked to have a useful conclusion, suggesting the best CK2 antibodies in relation to their different applications.

Author Response: We understand this concern and it is why we have chosen to submit the antibody characterization data in the format of Data Notes. As explained in the F1000 article guidelines, Data Notes do not include result analyses or conclusions. Furthermore, the authors prefer to remain unbiased while being transparent with the results obtained from the experiments. To provide context for readers who do not have experience analyzing results from such characterization methods utilizing knockdown lines as controls, we have included a subsequent Table to demonstrate how one can identify successful vs unsuccessful antibodies in each application. Please refer to Table 3 in the updated submission.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 05 Sep 2024

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

05 Sep 2024

Author Response

Thank you to Odile Filhol and Catherine Pillet for your thorough analysis of our manuscript presenting antibody characterization data for ten commercial CSNK2A1 antibodies to the scientific community. We hope ... Continue reading Thank you to Odile Filhol and Catherine Pillet for your thorough analysis of our manuscript presenting antibody characterization data for ten commercial CSNK2A1 antibodies to the scientific community. We hope our response to your comments and concerns clarify any misinterpretations and questions you may have previously had. Additionally, the authors have revisited the initial manuscript and made necessary changes to include a detailed methods section as to ensure the procedure is reproducible. As well, a limitation section has been included to address concerns regarding the nature of the platform and inherent limitations associated with it. Please refer to version 2.

Major points:
The major concern for me, as a specialist of CK2, is that there is not at least, a comment on the specificity of the antibodies to recognize the CSNK2A2 isoform, that is highly homolog to CSNK2A1. Does the HAP1 cell line used by the authors, express this isoform?

Author Response: Based on the characterization table provided by Depmap, HAP1 expresses the CSNK2A2 isoform at 5.8 log₂(TPM+1). That being said, this study is shared with the scientific community to help researchers find selective antibodies, already commercially available, for CSNK2A1. That is why a CSNK2A1 KO cell line was selected. To test the ability of the antibodies to recognize the isoform, we would need to test them in a CSNK2A2 KO line.

In the immunoprecipitation assays illustrated in Figure 2, 2 µg of each antibody was used. How did you decide to select 2 µg of antibody to coupled them on beads? However, either in Ponceau red staining or using the same antibody for western blot detection, for some of them, the heavy chain antibody is not visible. This is probably due to the low binding of the antibody. Could you please comment on that in the manuscript?

Author Response: The amount of 2 µg is selected based on previous experience with trial and error. 1ug was tested in the past and optimized to 2 µg to limit cross reactivity in the IP lane when performing the western blot for IP.

As for the ponceau staining, the initial concentrations provided by the manufacturers are followed when adding the 2 µg of antibody. In some cases, adhering to the manufacturers antibody concentrations can result in concentrations that are highly diluted. For example, the 2656 antibody from Cell Signalling Technology sis provided at a concentration of 0.03 µg/ul. Given this low concentration, the amount of antibody is very minimal preventing the heavy chain from being visible.

In Figure 3, could you show a least one picture representative of the HAP1 WT (green) and CSNK2A1 KO (far red) cells that allow identifying them.

Author Response: All of the underlying data is available in the Dataset, referenced in this manuscript and available on the YCharOS zenodo community. Please refer to the following link: https://doi.org/10.5281/zenodo.11078556 . The addition of Table 3 should also clarify how the WT and KO cells can be identified.

Minor points:
In table 2, stars in the second column are not useful since the information is provided in column 5. Stars and the corresponding legend should be removed.

Author Response: The stars are used outside throughout the report to highlight the renewable antibodies while also allowing the readers to remember the clonality of each antibody without having to refer back to Table 2.

In Figure 1 and 3, the antibody dilution should be indicated together with the name of each antibody.

Author Response: The antibody dilutions are indicated with the catalog number of each antibody in the figure legends.

Except the points raised in the comments above, the results are well presented.
However, I would have liked to have a useful conclusion, suggesting the best CK2 antibodies in relation to their different applications.

Author Response: We understand this concern and it is why we have chosen to submit the antibody characterization data in the format of Data Notes. As explained in the F1000 article guidelines, Data Notes do not include result analyses or conclusions. Furthermore, the authors prefer to remain unbiased while being transparent with the results obtained from the experiments. To provide context for readers who do not have experience analyzing results from such characterization methods utilizing knockdown lines as controls, we have included a subsequent Table to demonstrate how one can identify successful vs unsuccessful antibodies in each application. Please refer to Table 3 in the updated submission.
Thank you to Odile Filhol and Catherine Pillet for your thorough analysis of our manuscript presenting antibody characterization data for ten commercial CSNK2A1 antibodies to the scientific community. We hope our response to your comments and concerns clarify any misinterpretations and questions you may have previously had. Additionally, the authors have revisited the initial manuscript and made necessary changes to include a detailed methods section as to ensure the procedure is reproducible. As well, a limitation section has been included to address concerns regarding the nature of the platform and inherent limitations associated with it. Please refer to version 2.

Major points:
The major concern for me, as a specialist of CK2, is that there is not at least, a comment on the specificity of the antibodies to recognize the CSNK2A2 isoform, that is highly homolog to CSNK2A1. Does the HAP1 cell line used by the authors, express this isoform?

Author Response: Based on the characterization table provided by Depmap, HAP1 expresses the CSNK2A2 isoform at 5.8 log₂(TPM+1). That being said, this study is shared with the scientific community to help researchers find selective antibodies, already commercially available, for CSNK2A1. That is why a CSNK2A1 KO cell line was selected. To test the ability of the antibodies to recognize the isoform, we would need to test them in a CSNK2A2 KO line.

In the immunoprecipitation assays illustrated in Figure 2, 2 µg of each antibody was used. How did you decide to select 2 µg of antibody to coupled them on beads? However, either in Ponceau red staining or using the same antibody for western blot detection, for some of them, the heavy chain antibody is not visible. This is probably due to the low binding of the antibody. Could you please comment on that in the manuscript?

Author Response: The amount of 2 µg is selected based on previous experience with trial and error. 1ug was tested in the past and optimized to 2 µg to limit cross reactivity in the IP lane when performing the western blot for IP.

As for the ponceau staining, the initial concentrations provided by the manufacturers are followed when adding the 2 µg of antibody. In some cases, adhering to the manufacturers antibody concentrations can result in concentrations that are highly diluted. For example, the 2656 antibody from Cell Signalling Technology sis provided at a concentration of 0.03 µg/ul. Given this low concentration, the amount of antibody is very minimal preventing the heavy chain from being visible.

In Figure 3, could you show a least one picture representative of the HAP1 WT (green) and CSNK2A1 KO (far red) cells that allow identifying them.

Author Response: All of the underlying data is available in the Dataset, referenced in this manuscript and available on the YCharOS zenodo community. Please refer to the following link: https://doi.org/10.5281/zenodo.11078556 . The addition of Table 3 should also clarify how the WT and KO cells can be identified.

Minor points:
In table 2, stars in the second column are not useful since the information is provided in column 5. Stars and the corresponding legend should be removed.

Author Response: The stars are used outside throughout the report to highlight the renewable antibodies while also allowing the readers to remember the clonality of each antibody without having to refer back to Table 2.

In Figure 1 and 3, the antibody dilution should be indicated together with the name of each antibody.

Author Response: The antibody dilutions are indicated with the catalog number of each antibody in the figure legends.

Except the points raised in the comments above, the results are well presented.
However, I would have liked to have a useful conclusion, suggesting the best CK2 antibodies in relation to their different applications.

Author Response: We understand this concern and it is why we have chosen to submit the antibody characterization data in the format of Data Notes. As explained in the F1000 article guidelines, Data Notes do not include result analyses or conclusions. Furthermore, the authors prefer to remain unbiased while being transparent with the results obtained from the experiments. To provide context for readers who do not have experience analyzing results from such characterization methods utilizing knockdown lines as controls, we have included a subsequent Table to demonstrate how one can identify successful vs unsuccessful antibodies in each application. Please refer to Table 3 in the updated submission.
Competing Interests: No competing interests were disclosed. Close
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Version 2

VERSION 2 PUBLISHED 09 Jul 2024

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Odile Filhol, Université Grenoble-Alpes, Grenoble-Alpes, France

Catherine Pillet, French Alternative Energies and Atomic Energy Commission, Grenoble, France
David Litchfield, Western University, London, Canada
Miwako Homma, Fukushima Medical University School of Medicine, Fukushima, Japan

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10 Oct 2024 | for Version 2

David Litchfield, Western University, London, Ontario, Canada

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Approved

Based on the revisions that have been made, particularly the inclusion of limitations that explicitly acknowledge the existence of CSNK2A2 (a closely related isoform of CSNK2A1), I am satisfied that the Data Note is now suitable for indexing. Overall, the authors’ systematic evaluation of commercially available antibodies to detect the protein encoded by CSNK2A1 is an important contribution to the field.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Areas of expertise include signal transduction, protein kinases, kinase inhibitors and phosphoproteomics. The CK2 family of protein kinases (including CSNK2A1 and CSNK2A2) have represented a central focus of our research.

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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27 Aug 2024 | for Version 1

Miwako Homma, Fukushima Medical University School of Medicine, Fukushima, Japan

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Approved With Reservations

Antibody evaluations discussed in this manuscript are only comparative observations, and titers of individual antibodies are not quantified. As shown in Figure 1, only one concentration of each antibody diluent was selected, according to the supplier's recommendations. Affinity binding properties are generally verified quantitatively by ELISA, along with Western blotting, in which the intensity of each antibody to the target protein, in this case, purified CSNK2A1, can be calculated by stepwise dilution. I recommend that the authors compare each antibody at the concentration that maximizes performance and that blots be further quantified. It is interesting to note that there are few commercial antibodies that can immunoprecipitate the target molecule, CSNK2A1. However, immunoprecipitation was performed using only one concentration of the indicated antibodies.
Although CSNK2A1 (CK2) and CSNK2A2 (CK2’) are encoded at different loci, the amino acid sequences are very similar. Therefore, it is necessary to show that these antibodies are specific for CSNK2A1(CK2) and that they do not also recognize CSNK2A2 (CK2’). For this purpose, I recommend using cells with CSNK2A1 genetically knocked-out (ko) as a control, to show that antibody responses are not observed when only endogenous CANK2A2 is present, or when CSNK2A2 is expressed as a tagged cDNA in ko cells. Alternatively, antibody performance should be validated using human CSNK2A1 or CSNK2A2 recombinant proteins.

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

Intracellular signal transduction, protein kinases, CK2, phospho-proteomics, CK2-ChIP-Seq, RNA-Seq, epigenetics, and prognostic biomarker of cancer.

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02 Aug 2024 | for Version 1

David Litchfield, Western University, London, Ontario, Canada

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Approved With Reservations

Considering that the gene product encoded by CSNK2A1 is referred to by a variety of names in the published literature in addition to terms adopted by UniProt, it may be beneficial to include additional terms (including CK2alpha and/or CK2α as well as CKIIalpha or CKIIα that are commonly used in the literature). This suggestion is intended to maximize visibility of this article using different search engines.
The CSNK2A1 and CSNK2A2 genes encode proteins with very similar enzymatic characteristics and a very high degree of sequence similarity especially within their catalytic kinase domains. The main differences between the gene products encoded by CSNK2A1 and CSNK2A2 reside within their distinct C-terminal domains that extend beyond their kinase domains. In reviewing the technical information for some of the antibodies that was available on vendor websites, which in some cases was very limited due to proprietary considerations, it was not entirely clear to me whether some of these antibodies (particularly polyclonal antibodies and/or antibodies raised against recombinant proteins encompassing regions of very high similarity between CSNK2A1 and CSNK2A2) would also detect CSNK2A2 (ie. CK2α’). Overall, although it does not appear that CSNK2A2 (which represents a protein that is 41 amino acids shorter than the protein encoded by CSNK2A1) is detected by any of the antibodies in Western blots, I believe it would be beneficial to make direct reference to CSNK2A2 in this article and highlight this issue as a potential limitation and/or issue that warrants consideration. This issue would be particularly pertinent for studies to be performed in the absence of the paired cell lines (i.e., parental and knockout) as used in this report.

Minor suggestions.

Introduction (line 1) – need to add “kinase” following “serine/threonine” (either to replace “enzyme” or in addition to including “enzyme”)
Introduction (line 2). I would recommend replacing “essential” with less definitive terminology (potentially “with essential roles in”) since CSNK2A1 may not be universally involved in all of the listed processes.
Results and Discussion; Last sentence of 1^st paragraph and 1^st line of 2^nd paragraph; should be CSNK2A1 (instead of CSNK2A) – especially considering the existence of CSNK2A2 as noted above.
Results and Discussion. 2^rd paragraph. “were separated on SDS-PAGE” instead of “ran on SDS-PAGE”
Results and Discussion. 3^rd paragraph. “equal proportions” rather than “equal amounts”.

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

Respond to this report

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Author Response

05 Sep 2024

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

Thank you to David Litchfield for reviewing this manuscript. We hope our response to your comments and concerns clarify any misinterpretations and questions you may have previously had. Additionally, the authors have revisited the initial manuscript, including a detailed methods section to ensure the procedure is reproducible. As well, a limitation section has been included to address concerns regarding the inherent limitations of the overall platform as well as when evaluating CSNK2A1 antibodies. A third table has been included to help guide readers interpret the results and select high-performing antibodies, which is the aim of this study. Please refer to version 2.

Considering that the gene product encoded by CSNK2A1 is referred to by a variety of names in the published literature in addition to terms adopted by UniProt, it may be beneficial to include additional terms (including CK2alpha and/or CK2α as well as CKIIalpha or CKIIα that are commonly used in the literature). This suggestion is intended to maximize visibility of this article using different search engines.

Thank you for this suggestion, we are always looking to broaden our audience to increase end-user accessibility of our characterization data to enable researchers to select high-quality antibodies for their research. To this end, we have included the suggested terms to our list of keywords. Please refer to version 2.

2. The CSNK2A1 and CSNK2A2 genes encode proteins with very similar enzymatic characteristics and a very high degree of sequence similarity especially within their catalytic kinase domains. The main differences between the gene products encoded by CSNK2A1 and CSNK2A2 reside within their distinct C-terminal domains that extend beyond their kinase domains. In reviewing the technical information for some of the antibodies that was available on vendor websites, which in some cases was very limited due to proprietary considerations, it was not entirely clear to me whether some of these antibodies (particularly polyclonal antibodies and/or antibodies raised against recombinant proteins encompassing regions of very high similarity between CSNK2A1 and CSNK2A2) would also detect CSNK2A2 (ie. CK2α’). Overall, although it does not appear that CSNK2A2 (which represents a protein that is 41 amino acids shorter than the protein encoded by CSNK2A1) is detected by any of the antibodies in Western blots, I believe it would be beneficial to make direct reference to CSNK2A2 in this article and highlight this issue as a potential limitation and/or issue that warrants consideration. This issue would be particularly pertinent for studies to be performed in the absence of the paired cell lines (i.e., parental and knockout) as used in this report.

The authors are aware of the potential cross reactivity of the antibodies with CSNK2A2. The advantage of using genetic strategies (WT vs KO) for characterization is that we are able to detect whether the antibodies can or cannot target CSNK2A1 by assessing whether the signal is lost in the KO line. That being said, transparency and reproducibility is at the core of our initiative’s values which is why we have mentioned this factor in the limitations section, included in version 2 of the manuscript.

Minor suggestions.

Introduction (line 1) – need to add “kinase” following “serine/threonine” (either to replace “enzyme” or in addition to including “enzyme”)
Introduction (line 2). I would recommend replacing “essential” with less definitive terminology (potentially “with essential roles in”) since CSNK2A1 may not be universally involved in all of the listed processes.
Results and Discussion; Last sentence of 1^st paragraph and 1^stline of 2^nd paragraph; should be CSNK2A1 (instead of CSNK2A) – especially considering the existence of CSNK2A2 as noted above.
Results and Discussion. 2^rdparagraph. “were separated on SDS-PAGE” instead of “ran on SDS-PAGE”
Results and Discussion. 3^rdparagraph. “equal proportions” rather than “equal amounts”.

All of these minor changes have been attended to in version 2 of the manuscript which has been submitted.

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Competing Interests

No competing interests were disclosed.

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Reviewer Report

15 Views

01 Aug 2024 | for Version 1

Odile Filhol, Université Grenoble-Alpes, Grenoble-Alpes, France

Catherine Pillet, French Alternative Energies and Atomic Energy Commission, Grenoble, France

15 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

Cell biology and protein kinase CK2 specialist.

Respond to this report

Responses (1)

Author Response

05 Sep 2024

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

Thank you to Odile Filhol and Catherine Pillet for your thorough analysis of our manuscript presenting antibody characterization data for ten commercial CSNK2A1 antibodies to the scientific community. We hope our response to your comments and concerns clarify any misinterpretations and questions you may have previously had. Additionally, the authors have revisited the initial manuscript and made necessary changes to include a detailed methods section as to ensure the procedure is reproducible. As well, a limitation section has been included to address concerns regarding the nature of the platform and inherent limitations associated with it. Please refer to version 2.

Major points:
The major concern for me, as a specialist of CK2, is that there is not at least, a comment on the specificity of the antibodies to recognize the CSNK2A2 isoform, that is highly homolog to CSNK2A1. Does the HAP1 cell line used by the authors, express this isoform?

Author Response: Based on the characterization table provided by Depmap, HAP1 expresses the CSNK2A2 isoform at 5.8 log₂(TPM+1). That being said, this study is shared with the scientific community to help researchers find selective antibodies, already commercially available, for CSNK2A1. That is why a CSNK2A1 KO cell line was selected. To test the ability of the antibodies to recognize the isoform, we would need to test them in a CSNK2A2 KO line.

In the immunoprecipitation assays illustrated in Figure 2, 2 µg of each antibody was used. How did you decide to select 2 µg of antibody to coupled them on beads? However, either in Ponceau red staining or using the same antibody for western blot detection, for some of them, the heavy chain antibody is not visible. This is probably due to the low binding of the antibody. Could you please comment on that in the manuscript?

Author Response: The amount of 2 µg is selected based on previous experience with trial and error. 1ug was tested in the past and optimized to 2 µg to limit cross reactivity in the IP lane when performing the western blot for IP.

As for the ponceau staining, the initial concentrations provided by the manufacturers are followed when adding the 2 µg of antibody. In some cases, adhering to the manufacturers antibody concentrations can result in concentrations that are highly diluted. For example, the 2656 antibody from Cell Signalling Technology sis provided at a concentration of 0.03 µg/ul. Given this low concentration, the amount of antibody is very minimal preventing the heavy chain from being visible.

In Figure 3, could you show a least one picture representative of the HAP1 WT (green) and CSNK2A1 KO (far red) cells that allow identifying them.

Author Response: All of the underlying data is available in the Dataset, referenced in this manuscript and available on the YCharOS zenodo community. Please refer to the following link: https://doi.org/10.5281/zenodo.11078556 . The addition of Table 3 should also clarify how the WT and KO cells can be identified.

Minor points:
In table 2, stars in the second column are not useful since the information is provided in column 5. Stars and the corresponding legend should be removed.

Author Response: The stars are used outside throughout the report to highlight the renewable antibodies while also allowing the readers to remember the clonality of each antibody without having to refer back to Table 2.

In Figure 1 and 3, the antibody dilution should be indicated together with the name of each antibody.

Author Response: The antibody dilutions are indicated with the catalog number of each antibody in the figure legends.

Except the points raised in the comments above, the results are well presented.
However, I would have liked to have a useful conclusion, suggesting the best CK2 antibodies in relation to their different applications.

Author Response: We understand this concern and it is why we have chosen to submit the antibody characterization data in the format of Data Notes. As explained in the F1000 article guidelines, Data Notes do not include result analyses or conclusions. Furthermore, the authors prefer to remain unbiased while being transparent with the results obtained from the experiments. To provide context for readers who do not have experience analyzing results from such characterization methods utilizing knockdown lines as controls, we have included a subsequent Table to demonstrate how one can identify successful vs unsuccessful antibodies in each application. Please refer to Table 3 in the updated submission.

View more View less

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 CSNK2A1 (UniProt ID: P68400) for use in western blot, immunoprecipitation and immunofluorescence

Abstract

Keywords

Introduction

Results and discussion

Table 1. Summary of the cell lines used.

Table 2. Summary of the CSNK2A1 antibodies tested.

Figure 1. CSNK2A1 antibody screening by western blot.

Figure 2. CSNK2A1 antibody screening by immunoprecipitation.

Figure 3. CSNK2A1 antibody screening by immunofluorescence.

Methods

Antibodies and cell lines used

Data availability

Underlying data

Acknowledgment

References

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