CSNK2A2 Antibody

What is CSNK2A2 and what cellular functions does it regulate?

CSNK2A2, also known as casein kinase II alpha prime (CK2α'), is a catalytic subunit of protein kinase CK2 that plays crucial roles in signaling pathways regulating cell cycle progression and survival. This protein kinase is involved in various cellular processes including cell growth, proliferation, and apoptosis. CSNK2A2 functions as a key player in multiple signaling pathways and its dysregulation has been linked to various diseases, including cancer and neurodegenerative disorders, making it a promising therapeutic target .

The full amino acid sequence of human CSNK2A2 includes 350 amino acids as follows: MPGP AAGS RARV YAEV NSLR SREY WDYE AHVP SWGN QDDY QLVR KLGR GKYS EVFE AINI TNNE RVVV KILK PVKK KKIK REVK ILEN LRGG TNII KLID TVKD PVSK TPAL VFEY INNT DFKQ LYQI LTDF DIRF YMYE LLKA LDYC HSKG IMHR DVKP HNVM IDHQ QKKL RLID WGLA EFYH PAQE YNVR VASR YFKG PELL VDYQ MYDY SLDM WSLG CMLA SMIF RREP FFHG QDNY DQLV RIAK VLGT EELY GYLK KYHI DLDP HFND ILGQ HSRK RWEN FIHS ENRH LVSP EALD LLDK LLRY DHQQ RLTA KEAM EHPY FYPV VKEQ SQPC ADNA VLSS GLTA AR .

How can researchers differentiate between CSNK2A1 and CSNK2A2 in experimental settings?

Differentiating between the two catalytic subunits of CK2 (CSNK2A1/CK2α and CSNK2A2/CK2α') requires specific antibodies that can recognize their distinct C-terminal regions. While some antibodies can detect both catalytic subunits, researchers should use antibodies specifically raised against unique regions of each subunit for differential detection.

For specific detection of CSNK2A2, researchers can use antibodies raised against the C-terminal region of CSNK2A2. Some commercial antibodies, like the one mentioned in the search results, have been designed to specifically target CSNK2A2 . Alternatively, immunoprecipitation techniques using specific antibodies followed by mass spectrometry can help distinguish between the two proteins based on their unique peptide sequences.

When using Western blot for differentiation, careful selection of loading controls and comparison of molecular weights (CSNK2A1 and CSNK2A2 have slightly different molecular weights) can provide additional confirmation of specificity .

What are the common applications for CSNK2A2 antibodies in research?

CSNK2A2 antibodies have multiple applications in research settings:

• Western Blotting (WB): The primary application for detecting and quantifying CSNK2A2 protein expression in cell and tissue lysates. Typically used at dilutions of 1:500 - 1:2000 .

• ELISA: For quantitative detection of CSNK2A2 in solution .

• Immunoprecipitation (IP): For isolation of CSNK2A2 and its interacting partners from cell lysates, often used in combination with kinase assays .

• Intracellular Staining: For monitoring protein expression changes upon cellular stimulation in a time-dependent manner .

• Phosphorylation Studies: For identifying and validating CSNK2A2 substrates in combination with phospho-specific antibodies .

These applications enable researchers to study CSNK2A2's expression, regulation, and function in various biological contexts, particularly in cancer research and other disease models.

How should researchers validate the specificity of CSNK2A2 antibodies?

Validating CSNK2A2 antibody specificity requires multiple complementary approaches:

• Positive and Negative Controls: Use cell lines with known CSNK2A2 expression (293T, HeLa, K-562 as positive controls) . Include samples where CSNK2A2 has been knocked down or knocked out as negative controls.

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide before immunoblotting. Specific binding should be blocked by the peptide.

• Cross-Reactivity Testing: Test against recombinant CSNK2A1 and CSNK2A2 proteins to ensure the antibody specifically recognizes CSNK2A2 with minimal cross-reactivity.

• Multiple Antibody Validation: Compare results using antibodies from different sources or those recognizing different epitopes of CSNK2A2.

• Molecular Weight Verification: Confirm that the detected band appears at the expected molecular weight for CSNK2A2.

• Phosphatase Treatment Control: Include λ-phosphatase treated samples as controls when using phospho-specific antibodies to ensure specificity for the phosphorylated form .

These steps collectively ensure that experimental observations truly reflect CSNK2A2-specific signals rather than artifacts or cross-reactivity with related proteins.

What are the recommended protocols for CSNK2A2 detection in Western blot experiments?

For optimal Western blot detection of CSNK2A2, researchers should follow these procedural recommendations:

• Sample Preparation:

  • Lyse B-cells or other target cells in RIPA buffer with protease inhibitors

  • Include phosphatase inhibitors if phosphorylation status is important

  • Determine protein concentration using standard methods (Bradford, BCA)

• Gel Electrophoresis and Transfer:

  • Separate 20-40 μg of protein on 10-12% SDS-PAGE

  • Transfer to nitrocellulose membrane at 100V for 1 hour or 30V overnight

• Blocking and Antibody Incubation:

  • Block membrane in 3-5% BSA in TBST (0.05% Tween 20) for 1 hour at room temperature

  • Incubate with CSNK2A2 primary antibody at 1:500 - 1:2000 dilution in 3% BSA in TBST overnight at 4°C

  • Wash 3× with TBST, 5 minutes each

  • Incubate with appropriate secondary antibody in 1% BSA in TBST for 1 hour at room temperature

  • Wash 3× with TBST, 5 minutes each

• Detection and Controls:

  • Use β-Actin or GAPDH as loading controls

  • Include positive control lysates from 293T, HeLa, or K-562 cells

• Recommended Dilutions:

  • CSNK2A2 Polyclonal Antibody: 1:500 - 1:2000

  • β-Actin: 1:5000 - 1:10000

  • Secondary antibodies: 1:5000 - 1:10000

This standardized protocol helps ensure consistent and specific detection of CSNK2A2 in Western blot experiments.

What techniques can be used to study changes in CSNK2A2 expression during cell differentiation?

Researchers can employ several techniques to monitor CSNK2A2 expression changes during cell differentiation:

• Western Blotting: The most common method for tracking protein expression changes over time. Collect cells at different time points during differentiation and analyze CSNK2A2 levels using specific antibodies .

• Intracellular Flow Cytometry: Allows quantitative single-cell analysis of CSNK2A2 expression. This technique revealed time-dependent increases in CK2α protein expression upon stimulation with LPS in B-cells .

• Real-time PCR: Monitor changes in Csnk2a2 mRNA expression during differentiation to correlate with protein levels.

• Immunofluorescence Microscopy: Examine changes in both expression levels and subcellular localization during differentiation.

• CK2 Kinase Activity Assays: Measure functional changes in CK2 activity using commercial kits like the CycLex CK2 Assay/Inhibitor Screening Kit. This involves immunoprecipitating both catalytic subunits (CK2α and CK2α') and assessing their kinase activity .

For B-cell studies specifically, researchers observed that upon stimulation with LPS (T-cell independent stimulus) or CD40L plus IL-4 (T-cell dependent stimuli), expression of CK2α, CK2β, and CK2α' was strongly induced, indicating their importance in B-cell activation and differentiation .

What strategies can be employed to identify and validate novel CSNK2A2 substrates?

Identifying and validating novel CSNK2A2 substrates requires sophisticated methodological approaches:

• Chemical Genetics Combined with Phosphoproteomics: This strategy involves:

  • Creating inhibitor-resistant CSNK2A2 mutants (e.g., triple mutant V66A/H160D/I174A)

  • Treating cells with CSNK2 inhibitors like CX-4945 to inhibit endogenous CSNK2 activity

  • Expressing the inhibitor-resistant mutant to maintain phosphorylation of CSNK2 substrates

  • Using quantitative phosphoproteomics (SILAC) to identify maintained phosphorylation sites

• Phospho-Motif Antibodies: Use antibodies that recognize the CSNK2 consensus phosphorylation motif (pS/pTDXE) to identify potential substrates .

• Phospho-specific Antibody Development: Develop antibodies against putative CSNK2-dependent phosphorylation sites for validation, as demonstrated with EIF2S2 pS2, CSNK2B pS2/3/8, LIG1 pS36, and other sites .

• In Vitro Kinase Assays: Purify CSNK2A2 and test its ability to phosphorylate candidate substrates in vitro, followed by mass spectrometry to identify phosphorylation sites.

• Validation Using CSNK2 Inhibitors: Treat cells with selective CSNK2 inhibitors like CX-4945 or Inhibitor 8 and monitor changes in substrate phosphorylation. The most potent inhibitors in cellular context were identified as Inhibitor 8 and CX-4945 .

These approaches collectively provide robust identification and validation of CSNK2A2 substrates, contributing to our understanding of CSNK2A2's role in signaling networks.

How can phosphoproteomic approaches be optimized for CSNK2A2 research?

Optimizing phosphoproteomic approaches for CSNK2A2 research requires careful experimental design:

• Triple SILAC Quantitative Phosphoproteomics: Implementation of triple SILAC labeling allows comparison of multiple conditions simultaneously:

  • Light label: Control condition

  • Medium label: CSNK2 inhibitor treatment

  • Heavy label: CSNK2 inhibitor + expression of inhibitor-resistant CSNK2A2

• Phosphopeptide Enrichment Strategies:

  • TiO2 chromatography for global phosphopeptide enrichment

  • Phospho-motif antibody immunoprecipitation targeting CSNK2 consensus motifs (pS/pTDXE)

  • Sequential enrichment strategies to improve coverage of low-abundance phosphopeptides

• Bioinformatic Analysis Pipelines:

  • Motif analysis to identify enriched sequence motifs around phosphorylation sites

  • Network analysis to identify signaling pathways enriched for CSNK2A2 substrates

  • Integration with protein-protein interaction datasets

• Validation Approaches:

  • Development of phospho-specific antibodies against identified sites

  • Site-directed mutagenesis of putative phosphorylation sites

  • Functional assays to determine the biological relevance of identified phosphorylation events

• Inhibitor Controls:

  • Include multiple CSNK2 inhibitors with different chemical structures

  • Use inhibitor-resistant CSNK2A2 mutants as controls

  • Include phosphatase treatment controls to confirm phosphorylation-specific detection

This comprehensive approach maximizes the identification of genuine CSNK2A2 substrates while minimizing false positives.

What are the methodological considerations when studying CSNK2A2's role in disease models?

When investigating CSNK2A2's role in disease models such as cancer or neurodegenerative disorders, researchers should consider these methodological aspects:

• Model Selection:

  • Choose appropriate cell lines that reflect the disease context (e.g., 293T, HeLa, K-562 for cancer studies)

  • Consider patient-derived primary cells or organoid models for increased relevance

  • Validate findings in multiple independent model systems

• Inhibitor Strategy:

  • Consider the specificity profiles of CSNK2 inhibitors

  • CX-4945 has been evaluated against 238 kinases, with only 7 inhibited >90% at 500 nmol/L

  • CSNK2A1 and CSNK2A2 exhibited the lowest IC50 at 1 nmol/L CX-4945

  • Be aware of CSNK2-independent effects of inhibitors like CX-4945

• Genetic Approaches:

  • Use siRNA/shRNA for transient knockdown studies

  • Employ CRISPR/Cas9 for complete knockout or endogenous tagging

  • Consider isoform-specific approaches to distinguish CSNK2A1 vs. CSNK2A2 functions

• Pharmacological Validation:

  • Compare multiple CSNK2 inhibitors (e.g., TBB, TBBz, DMAT, Ellagic Acid, Quinalizarin, Resorufin, Inhibitor 8, CX-4945)

  • Test at multiple concentrations (1 μM, 10 μM, 50 μM)

  • Monitor inhibition using phospho-specific antibodies for known CSNK2 substrates

• Translational Considerations:

  • Correlate findings with clinical data when available

  • Consider the implications for therapeutic targeting

  • Acknowledge that CX-4945 is currently in clinical trials (NCT04663737, NCT04668209, NCT03904862)

These considerations help ensure that findings regarding CSNK2A2's role in disease are robust, reproducible, and potentially translatable to clinical applications.

How can researchers quantitatively measure CSNK2A2 kinase activity in cell or tissue samples?

Quantitative measurement of CSNK2A2 kinase activity requires specialized approaches:

• Commercial Kinase Assay Kits:

  • The CycLex CK2 Assay/Inhibitor Screening Kit allows for quantitative assessment of CK2 kinase activity

  • Protocol involves lysing cells, immunoprecipitating both catalytic subunits (CK2α and CK2α'), and measuring kinase activity according to manufacturer's instructions

• Immunoprecipitation-Based Kinase Assays:

  • Immunoprecipitate CSNK2A2 using specific antibodies

  • Incubate with recombinant substrate and ATP

  • Measure substrate phosphorylation using:
    a) Phospho-specific antibodies
    b) Radioactive ATP (³²P) incorporation
    c) Mass spectrometry

• Phospho-Substrate Monitoring:

  • Track phosphorylation of well-established CSNK2 substrates as proxies for activity

  • Use phospho-specific antibodies against sites like:
    a) CSNK2 pS/pTDXE motif
    b) XRCC1 pS518/T519/T523
    c) CDC37 pS13
    d) EIF2S2 pS2
    e) CSNK2B pS2/3/8 (autophosphorylation site)

• Inhibitor Dose-Response Analysis:

  • Treat cells with increasing concentrations of CSNK2 inhibitors

  • Quantify decreases in substrate phosphorylation

  • Calculate IC50 values in cellular context

• Data Normalization and Analysis:

  • Normalize to total protein levels (e.g., EIF2S2 pS2/total EIF2S2)

  • Define baseline activity (DMSO control) as 100%

  • Calculate relative activity after treatments

  • Use appropriate statistical methods to determine significance

This multi-faceted approach provides robust quantification of CSNK2A2 kinase activity under various experimental conditions.

What are the considerations when choosing between polyclonal and monoclonal CSNK2A2 antibodies?

Selecting between polyclonal and monoclonal CSNK2A2 antibodies requires careful consideration of research objectives:

For researchers studying CSNK2A2:

• Use polyclonal antibodies (like the CSNK2A2 Polyclonal Antibody mentioned ) when:

  • Maximum sensitivity is required

  • Detecting denatured proteins in Western blot

  • Working with low expression levels

• Use monoclonal antibodies when:

  • Absolute specificity between CSNK2A1 and CSNK2A2 is critical

  • Performing immunoprecipitation for substrate identification

  • Conducting long-term studies requiring consistent reagents

The search results reference a polyclonal antibody (CAB21368) generated in rabbits that has high specificity and sensitivity for human and mouse samples , making it suitable for Western blot experiments.

How can researchers troubleshoot inconsistent results when using CSNK2A2 antibodies?

When encountering inconsistent results with CSNK2A2 antibodies, researchers should systematically address potential issues:

• Antibody-Related Issues:

  • Storage and Handling: Ensure proper storage at recommended temperatures (typically -20°C) and avoid multiple freeze-thaw cycles

  • Dilution Optimization: Test multiple antibody dilutions (1:500 - 1:2000 for Western blot)

  • Batch Variation: Different lots may have varying specificity/sensitivity; consider obtaining a new batch

  • Antibody Age: Effectiveness may decrease over time; check expiration date

• Sample Preparation Factors:

  • Lysis Buffer Composition: Include appropriate protease and phosphatase inhibitors

  • Sample Degradation: Process samples quickly and keep on ice

  • Protein Concentration: Ensure consistent loading (20-40 μg typically)

  • Post-translational Modifications: Phosphorylation may affect antibody recognition

• Technical Considerations:

  • Blocking Optimization: Test different blocking agents (BSA vs. milk)

  • Incubation Conditions: Adjust temperature and duration (overnight at 4°C vs. room temperature)

  • Washing Steps: Increase number or duration of washes

  • Detection System: Compare chemiluminescence vs. fluorescence-based systems

• Controls and Validation:

  • Positive Controls: Include lysates from cells known to express CSNK2A2 (293T, HeLa, K-562)

  • Phosphatase Treatment: For phospho-specific antibodies, include λ-phosphatase treated samples

  • Recombinant Protein: Use purified CSNK2A2 as a positive control

  • Knockdown/Knockout Validation: Include CSNK2A2 depleted samples as negative controls

• Special Considerations for CSNK2A2:

  • Isoform Cross-Reactivity: Ensure the antibody doesn't cross-react with CSNK2A1

  • Cell Type Specificity: Expression levels vary between cell types

  • Stimulation Effects: B-cell stimulation with LPS, CD40L plus IL-4, or anti-IgM antibody plus IL-4 strongly induces CSNK2A2 expression

By systematically addressing these factors, researchers can identify and resolve the sources of inconsistency in CSNK2A2 antibody experiments.

How might CSNK2A2 antibodies contribute to therapeutic development for CSNK2-related diseases?

CSNK2A2 antibodies have significant potential to advance therapeutic development for CSNK2-related diseases through several research approaches:

• Target Validation and Biomarker Development:

  • CSNK2A2 antibodies can validate the protein's role in disease pathways

  • Monitor changes in CSNK2A2 expression or activity as potential biomarkers

  • Use phospho-specific antibodies to assess CSNK2 activity in patient samples

  • Correlation of CSNK2A2 levels with disease progression or treatment response

• Drug Discovery Support:

  • High-throughput screening of CSNK2 inhibitors using antibody-based assays

  • Evaluation of on-target effects of CX-4945 and other inhibitors in clinical trials

  • Distinguish CSNK2A1 vs. CSNK2A2-specific inhibition for targeted therapeutics

  • Assessment of inhibitor efficacy in cellular and animal models

• Mechanism-of-Action Studies:

  • Identify disease-specific CSNK2A2 substrates using the chemical genetics approach

  • Understand CSNK2A2's role in modulating signaling pathways in disease contexts

  • Differentiate between CSNK2-dependent and CSNK2-independent effects of inhibitors like CX-4945

  • Explore potential combination therapies targeting CSNK2A2 alongside other disease-relevant pathways

• Patient Stratification Strategies:

  • Identify patients likely to respond to CSNK2 inhibitors based on CSNK2A2 expression/activity

  • Develop companion diagnostics using CSNK2A2 antibodies for personalized medicine approaches

  • Monitor treatment efficacy through assessment of substrate phosphorylation

These research directions leverage CSNK2A2 antibodies not only as research tools but as critical components in translational pipelines toward developing effective therapies for cancer, neurodegenerative disorders, and other CSNK2-related pathologies.

What emerging technologies might enhance CSNK2A2 antibody-based research?

Several emerging technologies have the potential to revolutionize CSNK2A2 antibody-based research:

• Proximity Labeling Proteomics:

  • APEX2 or BioID fusion with CSNK2A2 to identify proximal proteins

  • TurboID for rapid biotin labeling of CSNK2A2 interaction partners

  • Integrate with phosphoproteomics to identify substrates in their native cellular environment

• Single-Cell Analysis Techniques:

  • Single-cell Western blotting for heterogeneity analysis of CSNK2A2 expression

  • Mass cytometry (CyTOF) with CSNK2A2 antibodies for high-dimensional profiling

  • Single-cell phosphoproteomics to correlate CSNK2A2 activity with cellular phenotypes

• Advanced Imaging Technologies:

  • Super-resolution microscopy to visualize CSNK2A2 subcellular localization

  • FRET-based biosensors to monitor CSNK2 activity in living cells

  • Expansion microscopy combined with CSNK2A2 antibodies for enhanced spatial resolution

• Antibody Engineering Approaches:

  • Nanobodies or single-domain antibodies against CSNK2A2 for improved penetration

  • Bispecific antibodies targeting CSNK2A2 and its substrates

  • Intrabodies for targeting CSNK2A2 in live cells

• Spatial Omics Integration:

  • Spatial transcriptomics combined with CSNK2A2 immunohistochemistry

  • Digital spatial profiling with CSNK2A2 antibodies

  • Imaging mass cytometry for spatial analysis of CSNK2A2 and its substrates

These technologies, when applied to CSNK2A2 research, promise to provide unprecedented insights into its spatial and temporal regulation, substrate specificity, and role in disease pathogenesis, ultimately accelerating therapeutic development for CSNK2-related disorders.