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. 2024 Sep 5;13:781. Originally published 2024 Jul 9. [Version 2] doi: 10.12688/f1000research.153243.2

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

Riham Ayoubi 1, Maryam Fotouhi 1, Charles Alende 1, Vera Ruíz Moleón 1, Kathleen Southern 1, Carl Laflamme 1,a; NeuroSGC/YCharOS/EDDU collaborative group; ABIF consortium
PMCID: PMC11472280  PMID: 39403680

Version Changes

Revised. Amendments from Version 1

To enhance transparency and reproducibility in the antibody characterization process, version 2 has been created. The methods section now provides a description of the experimental setup for each application in which the antibodies were tested. In the Results and Discussion section, an additional table has been added to guide viewers in assessing the antibody characterization results. Lastly, a Limitations section has been introduced to address the inherent limitations of the platform.

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

Introduction

Casein kinase II subunit alpha (CSNK2A1), encoded by the CSNK2A1 gene, is a catalytic subunit of the serine/threonine kinase, casein kinase 2; important 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 protein’s 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 log 2 (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 log 2 (TPM+1) RNA levels, which is above the average range of cancer cells analyzed. Parental and CSNK2A1 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
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For western blot experiments, WT and CSNK2A1 KO protein lysates were separated on SDS-PAGE, transferred 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
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Wb = western blot; IF = immunofluorescence; IP = immunoprecipitation.

*

Monoclonal antibody.

**

Recombinant antibody.

Figure 1. CSNK2A1 antibody screening by western blot.

Figure 1.

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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 ( Figure 1) was selected. Equal proportions 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.

Figure 2.

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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.

Figure 3.

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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. To guide the results assessment by the viewer for each corresponding application, Table 3 provides an illustration of the different scenarios an antibody can perform and the outcome interpreted in all three applications ( Table 3). 18 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.

Table 3. Illustrations to assess antibody performance in all three applications.

Western Blot Immunoprecipitation Immunofluorescence
graphic file with name f1000research-13-170469-i0000.jpg graphic file with name f1000research-13-170469-i0001.jpg graphic file with name f1000research-13-170469-i0002.jpg
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This table has been reproduced with permission from Ayoubi et al., Elife, 2023. 18 (CC BY license).

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. 28 , 29

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 Brief descriptions of the experimental setup used to carry out this study can be found below.

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 Peroxidase-conjugated goat anti-rabbit and anti-mouse are from Thermo Fisher Scientific (cat. number 65-6120 and 62-6520). Alexa-555-conjugated goat anti-rabbit and anti-mouse secondary antibodies are from Thermo Fisher Scientific (cat. number A21429 and A21424).

Antibody screening by western blot

HAP1 WT and CSNK2A1 KO (listed in Table 1) were collected in RIPA buffer (25mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) from Thermo Fisher Scientific (cat. number 89901) supplemented with 1x protease inhibitor cocktail mix (MilliporeSigma, cat. number P8340). Lysates were sonicated briefly and incubated 30 min on ice. Lysates were spun at ~110,000×g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and western blot. BLUelf prestained protein ladder from GeneDireX (cat. number PM008-0500) was used.

Western blots were performed with precast midi 4-20% Tris-Glycine polyacrylamide gels from Thermo Fisher Scientific (cat. number WXP42012BOX) ran with Tris/Glycine/SDS buffer from Bio-Rad (cat. number 1610772), loaded in Laemmli loading sample buffer from Thermo Fisher Scientific (cat. number AAJ61337AD) and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau S staining (Thermo Fisher Scientific, cat. number BP103-10) which is scanned to show together with individual western blot. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated overnight at 4°C with 5% milk in TBS with 0,1% Tween 20 (TBST) from Cell Signaling (cat. number 9997). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 μg/ml in TBST with 5% milk for 1 hr at room temperature followed by three washes with TBST. Membranes were incubated with Pierce ECL from Thermo Fisher Scientific (cat. number 32106) prior to detection with the iBright™ CL1500 Imaging System from Thermo Fisher Scientific (cat. number A44240).

Antibody screening by immunoprecipitation

Antibody-beads conjugates were prepared by adding 2 μg or 10 μl of antibody 2656 to 500 μl of Pierce IP Lysis Buffer from Thermo Fisher Scientific (cat. number 87788) in a microcentrifuge tube, together with with 30μl of Dynabeads protein A- (for rabbit antibodies) or protein G- (for mouse antibodies) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively). Tubes were rocked for ~1 hr at 4°C followed by two washes to remove unbound antibodies.

HAP1 WT were collected in Pierce IP buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) supplemented with protease inhibitor. Lysates were rocked 30 min at 4°C and spun at 110,000xg for 15 min at 4°C. 0.5 ml aliquots at 2.0 mg/ml of lysate were incubated with an antibody-bead conjugate for ~1 hr at 4°C. The unbound fractions were collected, and beads were subsequently washed three times with 1.0 ml of IP lysis buffer and processed for SDS-PAGE and western blot on precast midi 4-20% Tris-Glycine polyacrylamide gels. VeriBlot for IP Detection Reagent:HRP (Abcam, cat. number ab131366) was used as a secondary detection system at a concentration of 0.1 μg/ml.

Antibody screening by immunofluorescence

HAP1 WT and CSNK2A1 KO were labelled with a green and a far-red fluorescence dye, respectively. The fluorescent dyes used are from Thermo Fisher Scientific (cat. number C2925 and C34565). WT and KO cells were plated in a 96-well plate with optically clear flat-bottom (Perkin Elmer, cat. number 6055300) as a mosaic and incubated for 24 hrs in a cell culture incubator at 37 oC, 5% CO 2. Cells were fixed in 4% PFA (in PBS) for 15 min at room temperature and then washed 3 times with PBS. Cells were permeabilized in PBS with 0,1% Triton X-100 for 10 min at room temperature and blocked with PBS with 5% BSA, 5% goat serum and 0.01% Triton X-100 for 30 min at room temperature. Cells were incubated with IF buffer (PBS, 5% BSA, 0,01% Triton X-100) containing the primary Casein kinase II subunit alpha antibodies overnight at 4°C. Cells were then washed 3 × 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 μg/ml for 1 hr at room temperature with DAPI. Cells were washed 3 × 10 min with IF buffer and once with PBS.

Images were acquired on an ImageXpress micro confocal high-content microscopy system (Molecular Devices), using a 20x NA 0.95 water immersion objective and scientific CMOS cameras, equipped with 395, 475, 555 and 635 nm solid state LED lights (lumencor Aura III light engine) and bandpass filters to excite DAPI, Cellmask Green, Alexa-555 and Cellmask Red, respectively. Images had pixel sizes of 0.68 x 0.68 microns, and a z-interval of 4 microns. For analysis and visualization, shading correction (shade only) was carried out for all images. Then, maximum intensity projections were generated using 3 z-slices. Segmentation was carried out separately on maximum intensity projections of Cellmask channels using CellPose 1.0, and masks were used to generate outlines and for intensity quantification. 27 Figures were assembled with Adobe Illustrator.

Limitations

Inherent limitations are associated with the antibody characterization platform employed in this study. 11 The authors do not claim to have expertise in CSNK2A1, which is why a brief background of the protein’s function and relevance in disease is provided. Adopting an agnostic approach, the authors perform antibody-based applications and share the results openly, leaving the analysis and interpretation up to the readers.

One limitation that may arise in this particular study is the antibodies potential to cross-react with CSNK2A2. Although the commercial antibodies are marketed as targeting CSNK2A1 on their distinctive catalogs, the proprietary information is not always provided. That being said, applying the genetic strategy and testing the antibodies in WT and CSNK2A1 KO cell lines allows researchers to identify selective and renewable CSNK2A1 antibodies for their experimental needs.

For the YCharOS effort, experiments are not performed in replicates. The rationale behind this approach is related to the fact that the validation of the KO cell lines involves the use of multiple antibodies targeting various epitopes. Once a specific antibody is identified, it validates the protein expression of the intended target in the selected cell line at a concentration that is detectible by a suitable antibody and supports conclusions regarding the specificity of the other antibodies. All experiments are performed using master mixes, and meticulous attention is paid to sample preparation and experimental execution. In IF, the use of two different concentrations serves to evaluate antibody specificity and can aid in assessing assay reliability. In instances where antibodies yield no signal, a repeat experiment is conducted.

As comprehensive and standardized procedures are respected, any conclusions remain confined to the experimental conditions and cell line used for this study. The use of a signle cell line for evaluating antibody performance poses as a limitation, as factors such as target protein abundance significantly impact results. Additionally, the use of cancer cell lines containing gene mutations poses a potential challenge, as these mutations may be within the epitope coding sequence or other regions of the gene responsible for the intended target. Such alterations should impact the binding affinity of antibodies. This represents an inherent limitation of any approach that employs cancer cell lines.

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).

Funding Statement

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.

[version 2; peer review: 1 approved

Data availability

Underlying data

Zenodo: Antibody Characterization Report for CSNK2A1, doi.org/10.5281/zenodo.10818214. 28

Zenodo: Dataset for the CSNK2A1 antibody screening study, doi.org/10.5281/zenodo.11078556. 29

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

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F1000Res. 2024 Oct 10. doi: 10.5256/f1000research.170469.r321082

Reviewer response for version 2

David Litchfield 1

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.

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

Yes

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

Yes

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

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

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.

F1000Res. 2024 Aug 27. doi: 10.5256/f1000research.168109.r303889

Reviewer response for version 1

Miwako Homma 1

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.

  1. 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.

  2. 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.

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

Yes

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

Yes

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

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

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.

F1000Res. 2024 Aug 2. doi: 10.5256/f1000research.168109.r303880

Reviewer response for version 1

David Litchfield 1

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.

 

  1. 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.

  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. 

Minor suggestions.

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

  2. 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.   

  3. 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. 

  4. Results and Discussion.  2 rd paragraph.  “were separated on SDS-PAGE” instead of “ran on SDS-PAGE”

  5. Results and Discussion.  3 rd paragraph.  “equal proportions” rather than “equal amounts”. 

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

Yes

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

Yes

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

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

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, however I have significant reservations, as outlined above.

F1000Res. 2024 Aug 14.
Kathleen Southern 1

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.

 

  1. 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. 

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

  2. 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.   

  3. 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. 

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

  5. 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.

F1000Res. 2024 Aug 1. doi: 10.5256/f1000research.168109.r303886

Reviewer response for version 1

Odile Filhol 1, Catherine Pillet 2

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.

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

Yes

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

Yes

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

Partly

Are the protocols appropriate and is the work technically sound?

Yes

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.

F1000Res. 2024 Aug 14.
Kathleen Southern 1

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 log 2(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.

Associated Data

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

    Data Citations

    1. Ayoubi R, Laflamme C: Dataset for the Casein kinase II subunit alpha antibody screening study.[Dataset]. Zenodo. 2024. 10.5281/zenodo.11078556 [DOI]

    Data Availability Statement

    Underlying data

    Zenodo: Antibody Characterization Report for CSNK2A1, doi.org/10.5281/zenodo.10818214. 28

    Zenodo: Dataset for the CSNK2A1 antibody screening study, doi.org/10.5281/zenodo.11078556. 29

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

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