Keywords
UniProt ID P68400, CSNK2A1, Casein kinase II subunit alpha, antibody characterization, antibody validation, western blot, immunoprecipitation, immunofluorescence
This article is included in the YCharOS (Antibody Characterization through Open Science) gateway.
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.
UniProt ID P68400, CSNK2A1, Casein kinase II subunit alpha, antibody characterization, antibody validation, western blot, immunoprecipitation, immunofluorescence
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.
See the authors' detailed response to the review by Odile Filhol and Catherine Pillet
See the authors' detailed response to the review by David Litchfield
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
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 CSNK2A1 KO HAP1 cells were obtained from Horizon Discovery (Table 1).
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 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).
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 |
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).
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.
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.
Western Blot | Immunoprecipitation | Immunofluorescence |
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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
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.
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).
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-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.
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 37oC, 5% CO2. 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.
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.
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).
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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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.
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.
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.
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.
Alongside their report, reviewers assign a status to the article:
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Version 1 09 Jul 24 |
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Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. Consider the following examples, but note that this is not an exhaustive list:
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