CKL2 Antibody

What is Protein Kinase CK2 and why are antibodies against it important for research?

Protein kinase CK2 (formerly known as Casein Kinase 2) is a ubiquitously expressed, constitutively active serine/threonine kinase that typically forms tetrameric complexes consisting of two catalytic subunits (α and/or α') and two regulatory (β) subunits . CK2 phosphorylates multiple protein substrates and plays crucial roles in diverse cellular and biological processes .

Antibodies against CK2 are essential research tools because they enable:

• Detection and quantification of CK2 expression in different cell types and tissues

• Evaluation of CK2 as a potential therapeutic target in various diseases

• Investigation of CK2's involvement in signaling pathways, particularly in immune cell differentiation and cancer progression

• Assessment of CK2's interactions with other proteins through techniques like immunoprecipitation

What are the key CK2 subunits that antibodies typically target?

CK2 antibodies are designed to target specific subunits of the protein kinase complex:

• CK2α (CSNK2A1): The primary catalytic subunit with a molecular weight of approximately 42-47 kDa

• CK2α': An alternative catalytic subunit that can substitute for CK2α in some complexes

• CK2β: The regulatory subunit that modulates the activity and substrate specificity of the catalytic subunits

When selecting CK2 antibodies, researchers should consider which subunit is most relevant to their specific research question. For example, studies on B-cell development might focus on CK2α, as conditional deletion of this subunit impacts B-cell differentiation patterns .

How do I validate the specificity of a CK2 antibody for my research?

Antibody validation is crucial for ensuring experimental reliability. For CK2 antibodies, a comprehensive validation approach includes:

• Knockout validation: Compare antibody reactivity between wild-type cells and CK2 knockout cell lines. For example, the MAB7957 antibody shows specific binding to CK2α at ~47 kDa in parental HAP1 cells but no signal in CK2α knockout HAP1 cells .

• Western blot analysis: Verify that the antibody detects bands of the expected molecular weight (approximately 42-47 kDa for CK2α) .

• Immunocytochemistry with knockout controls: Verify antibody specificity in immunofluorescence applications using paired wild-type and knockout cells .

• Positive and negative tissue controls: Use tissues known to express or lack CK2 to confirm staining specificity .

• Cross-reactivity testing: Ensure the antibody doesn't react with closely related kinases or other proteins with similar epitopes .

How should I design experiments to detect changes in CK2 expression upon cellular activation?

When designing experiments to monitor CK2 expression changes during cellular activation:

• Time course analysis: As demonstrated with B-cell activation, CK2 subunit expression changes occur in a time-dependent manner. Monitor CK2α, CK2β, and CK2α' expression at multiple time points (e.g., 0, 24, 48, 72 hours) after stimulation .

• Multiple detection methods: Combine Western blotting with intracellular staining for flow cytometry to capture both population-level and single-cell expression changes .

• Parallel mRNA and protein analysis: Correlate protein expression changes with corresponding mRNA levels of Csnk2a1 (CK2α), Csnk2b1 (CK2β), and Csnk2a2 (CK2α') to determine if regulation occurs at the transcriptional level .

• Appropriate stimuli selection: For immune cells, include both T-cell-dependent stimuli (e.g., CD40L plus IL-4, anti-IgM antibody plus IL-4) and T-cell-independent stimuli (e.g., LPS) to comprehensively assess CK2 regulation .

• Controls: Include unstimulated cells at each time point to account for time-dependent changes unrelated to stimulation .

What are the optimal methods for assessing CK2 kinase activity using antibodies?

CK2 kinase activity can be effectively measured using antibody-based approaches:

• Immunoprecipitation followed by kinase assay:

  • Lyse cells in an appropriate buffer

  • Immunoprecipitate both catalytic subunits (CK2α and CK2α') using specific antibodies

  • Assess kinase activity of the immunoprecipitated complex using commercial kits like the CycLex CK2 Assay/Inhibitor Screening Kit

• Phospho-specific antibody detection:

  • Treat cells with CK2 activators or inhibitors

  • Perform Western blotting with antibodies that recognize CK2-specific phosphorylation sites on known substrates

  • Compare phosphorylation levels with total protein levels using subunit-specific antibodies

• In-cell activity assays:

  • Transfect cells with CK2 activity reporters

  • Treat with stimuli or inhibitors

  • Use immunofluorescence to correlate CK2 localization (detected by antibodies) with kinase activity

How can I effectively use CK2 antibodies in co-immunoprecipitation experiments to identify interaction partners?

For successful co-immunoprecipitation (co-IP) experiments with CK2 antibodies:

• Antibody selection: Choose antibodies specifically validated for immunoprecipitation applications. Some antibodies (like sc-373894) have been successfully used to immunoprecipitate CK2α and identify interactions with proteins like NF-κB, PTEN, and β-catenin.

• Lysis conditions: Use non-denaturing lysis buffers (e.g., RIPA buffer) that preserve protein-protein interactions while efficiently extracting CK2 complexes .

• Pre-clearing: Pre-clear lysates with appropriate control IgG and protein A/G beads to reduce non-specific binding.

• Cross-linking consideration: For transient or weak interactions, consider using chemical cross-linkers before cell lysis.

• Reciprocal co-IP: Confirm interactions by performing reciprocal co-IP experiments using antibodies against the potential interaction partners.

• Controls: Always include:

  • IgG control immunoprecipitation

  • Input sample (typically 5-10% of the lysate used for IP)

  • When possible, samples from cells with knocked-down or knocked-out CK2 expression

How can CK2 antibodies be used to investigate its role in immune cell differentiation and function?

CK2 antibodies enable sophisticated investigations into immune cell biology:

• B-cell differentiation studies:

  • Use CK2α antibodies to track expression changes during B-cell development stages

  • Compare CK2α levels between different B-cell subsets (transitional B-cells vs. marginal zone B-cells)

  • Correlate CK2α expression with BCR and Notch2 signaling markers

• T-cell polarization analysis:

  • Monitor CK2 expression during Th17/Th1 differentiation versus Treg generation

  • Use intracellular staining with CK2 antibodies combined with lineage-specific transcription factor antibodies

  • Correlate CK2 activity with cytokine production profiles

• Dendritic cell function:

  • Investigate CK2-mediated PD-L1 phosphorylation at Thr285/Thr290 using phospho-specific antibodies

  • Assess how CK2 inhibition affects PD-L1 stability and T-cell activation capacity

• Conditional knockout validation:

  • Confirm CK2 deletion efficiency in conditional knockout models (e.g., CD19-Cre × Csnk2a1^fl/fl^) using CK2α antibodies

  • Compare results from flow cytometry, Western blotting, and immunohistochemistry to ensure complete deletion

What approaches should I use to investigate CK2 expression in tumor samples and its correlation with clinical outcomes?

For investigating CK2 in tumor contexts:

How can I use CK2 antibodies to study post-translational modifications and their impact on protein function?

To investigate CK2-mediated post-translational modifications:

• Phospho-specific antibody approaches:

  • Use antibodies that specifically recognize CK2 phosphorylation motifs (S/T-X-X-E/D/pS)

  • Validate phosphorylation sites using phosphatase treatments and site-directed mutagenesis

  • Monitor changes in phosphorylation dynamics under different cellular conditions

• Proximity ligation assays (PLA):

  • Combine CK2 antibodies with antibodies against potential substrates

  • Visualize and quantify direct interactions in situ without disrupting cellular architecture

  • Compare interaction frequencies under different treatment conditions

• Mass spectrometry validation:

  • Immunoprecipitate CK2 and associated proteins using validated antibodies

  • Identify phosphorylation sites through mass spectrometry analysis

  • Confirm specific sites using phospho-specific antibodies in follow-up experiments

• Functional impact assessment:

  • Correlate changes in protein phosphorylation (detected by phospho-specific antibodies) with alterations in protein stability, localization, or activity

  • Use CK2 inhibitors to confirm the kinase-dependency of observed modifications

  • Compare wild-type and phospho-mutant proteins to establish functional consequences

What are the critical factors for optimizing Western blot protocols with CK2 antibodies?

For optimal Western blot results with CK2 antibodies:

• Sample preparation:

  • Use appropriate lysis buffers (e.g., RIPA buffer) with protease and phosphatase inhibitors

  • Maintain consistent protein loading (10-30 μg total protein per lane)

  • Include positive controls (recombinant CK2) and negative controls (CK2 knockout samples)

• Antibody selection and dilution:

  • Choose antibodies validated specifically for Western blot applications

  • Use recommended dilutions (typically 1:100-1:1000) and optimize if necessary

  • Consider antibody combinations to detect multiple CK2 subunits simultaneously

• Detection optimization:

  • For CK2α, expect bands at approximately 42-47 kDa

  • Use reducing conditions for most CK2 antibody applications

  • Verify specificity using knockout cell lines when available

• Troubleshooting considerations:

  • For weak signals, extend primary antibody incubation time (overnight at 4°C)

  • For high background, increase blocking time or adjust detergent concentration

  • For multiple bands, verify with knockout controls and consider using monoclonal antibodies for greater specificity

What are the best practices for immunofluorescence and immunohistochemistry with CK2 antibodies?

For optimal immunostaining with CK2 antibodies:

• Fixation and antigen retrieval:

  • For immunofluorescence on cultured cells, 4% paraformaldehyde fixation is typically effective

  • For paraffin-embedded tissues, heat-induced epitope retrieval methods are often necessary

  • Optimize antigen retrieval conditions (pH, buffer composition, duration) for each antibody

• Validation controls:

  • Use paired wild-type and CK2 knockout cells to confirm staining specificity

  • Include isotype controls to assess non-specific binding

  • Consider dual labeling with antibodies against different CK2 subunits or epitopes

• Signal detection and analysis:

  • For immunofluorescence, counterstain nuclei with DAPI for proper cellular context

  • For mixed cell populations, combine with lineage markers for accurate cell identification

  • Quantify staining intensity and subcellular localization using appropriate image analysis software

• Protocol optimization:

  • Determine optimal antibody concentration (starting with 1 μg/mL for most applications)

  • Test different permeabilization reagents (0.1-0.5% Triton X-100 vs. 0.1% saponin)

  • Evaluate background reduction strategies (e.g., serum blocking, BSA, milk proteins)

How should I approach antibody selection for studying CK2 in different model organisms?

When selecting CK2 antibodies for cross-species applications:

• Epitope conservation analysis:

  • Compare the amino acid sequence of the antibody's epitope across species

  • Select antibodies raised against highly conserved regions for cross-species application

  • Consider species-specific antibodies when studying regions with significant sequence divergence

• Validation in each model system:

  • Test antibody reactivity in each species before conducting full experiments

  • Use positive controls (tissues known to express CK2) from the target species

  • When available, use knockout/knockdown samples from each species as negative controls

• Species-specific considerations:

  • For mouse models: Many anti-human CK2 antibodies cross-react with mouse proteins due to high sequence conservation

  • For non-mammalian models: Validate antibody specificity using Western blot before immunostaining applications

  • For human clinical samples: Prioritize antibodies validated on human tissues with appropriate controls

• Reported cross-reactivity:

  • Some antibodies (like the polyclonal antibody BS-1005R) have confirmed reactivity across human, rat, and mouse samples

  • Compare staining patterns across species to ensure consistent target recognition

  • Consider raising custom antibodies for poorly conserved regions in non-traditional model organisms

How can I address common issues with background staining when using CK2 antibodies?

To minimize background and improve signal-to-noise ratio:

• Antibody-specific optimization:

  • Titrate primary antibody concentration to determine optimal working dilution

  • Test different incubation conditions (temperature, duration, buffer composition)

  • Compare monoclonal versus polyclonal antibodies for your specific application

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blocking buffers)

  • Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Consider adding detergents (0.1-0.3% Triton X-100 or Tween-20) to reduce non-specific binding

• Sample preparation refinement:

  • Ensure proper fixation without overfixation (which can increase background)

  • For tissues, optimize antigen retrieval protocols to improve signal-to-noise ratio

  • Use fresh reagents and filtered solutions to minimize artifacts

• Controls and countermeasures:

  • Include secondary-only controls to assess secondary antibody background

  • Use isotype controls matched to the primary antibody

  • Consider using knockout tissues/cells as definitive negative controls

  • When available, block with immunizing peptide to confirm specificity

How do I interpret conflicting results between different detection methods for CK2?

When facing inconsistent results across different techniques:

• Methodological considerations:

  • Different detection methods have varying sensitivities and limitations

  • Western blots detect denatured proteins while immunofluorescence observes proteins in their cellular context

  • Immunoprecipitation may detect protein complexes rather than individual subunits

• Systematic validation approach:

  • Verify antibody specificity using knockout controls in each detection method

  • Test multiple antibodies targeting different epitopes of the same protein

  • Consider protein conformation and accessibility of epitopes in each method

• Reconciliation strategies:

  • Use orthogonal techniques (e.g., mass spectrometry) to validate conflicting results

  • Evaluate whether discrepancies reflect true biological differences (e.g., cellular heterogeneity)

  • Consider post-translational modifications that might affect antibody recognition

• Data interpretation framework:

  • Prioritize results from methods with the most robust controls

  • Consider context-specific factors (cell type, activation state, environmental conditions)

  • Acknowledge limitations in the discussion of results

What factors should I consider when comparing CK2 antibody data across different research groups?

When comparing CK2 data from different studies:

• Antibody specifications:

  • Identify the exact antibody clone, vendor, and catalog number used

  • Compare the epitope regions targeted by different antibodies

  • Note whether antibodies target different CK2 subunits or isoforms

• Methodological variations:

  • Assess differences in experimental protocols (sample preparation, dilutions, detection methods)

  • Consider cell/tissue-specific factors that might influence antibody performance

  • Note variations in quantification and normalization approaches

• Model system differences:

  • Account for variations in cell lines, primary cells, or animal models used

  • Consider genetic background differences that might affect CK2 expression or function

  • Note treatments or stimulations that could alter CK2 levels or activity

• Reporting standards assessment:

  • Evaluate the comprehensiveness of antibody validation reported

  • Look for inclusion of appropriate positive and negative controls

  • Consider whether knockout validation was performed