CKA2 Antibody

What is CK2 (Casein Kinase 2) and why is it important in research?

CK2 is a ubiquitous and constitutively active serine/threonine protein kinase involved in various cellular functions. It typically forms tetrameric complexes consisting of two catalytic subunits (alpha and/or alpha') and two regulatory (beta) subunits . CK2 is significant in research because it participates in numerous signaling pathways that, when deregulated, can contribute to human pathologies . The alpha subunit (CK2α) is approximately 42-47 kDa in size as detected by Western blot and plays crucial roles in cellular processes including cell proliferation, apoptosis, and gene expression.

To study CK2α in experimental contexts, researchers should:

• Consider its constitutive activity when designing experiments

• Examine both nuclear and cytoplasmic localization, as CK2α has been detected in both compartments

• Account for its interaction with the regulatory beta subunit when studying its function

What applications can CK2α antibodies be used for in laboratory research?

CK2α antibodies have been validated for multiple research applications:

• Western Blotting: Successfully detects CK2α at approximately 42-47 kDa in human, mouse, and rat cell lines . Optimal dilution is typically 0.5-1 μg/mL .

• Immunocytochemistry/Immunofluorescence: CK2α antibodies can be used to visualize protein localization in fixed cells. Effective concentration is around 25 μg/mL, with typical incubation for 3 hours at room temperature .

• Immunohistochemistry: Detects CK2α in paraffin-embedded tissue sections at 5 μg/mL with overnight incubation at 4°C .

• Immunoprecipitation: Several clones produce antibodies that specifically recognize CK2β subunit and are suitable for immunoprecipitation experiments .

For optimal results, each researcher should perform titration experiments to determine the ideal antibody concentration for their specific experimental system and application.

How do I validate the specificity of a CK2α antibody?

Proper validation is critical for ensuring experimental reproducibility and reliable data interpretation:

• Knockout/Knockdown Controls: Use CK2α knockout cell lines as negative controls. The absence of signal in knockout lines confirms antibody specificity, as demonstrated with HAP1 CK2α knockout cells .

• Multiple Detection Methods: Verify specificity using different techniques (e.g., Western blot and immunocytochemistry) to ensure consistent recognition of the target.

• Cross-Species Reactivity Assessment: Test antibody against CK2α from different species if cross-reactivity is claimed. Some antibodies have confirmed reactivity with human, mouse, and rat CK2α .

• Multiple Antibody Comparison: Use different antibodies targeting distinct epitopes of CK2α to confirm results.

• Recombinant Protein Controls: Include purified recombinant CK2α protein as a positive control in Western blots.

What are the differences between monoclonal and polyclonal CK2 antibodies?

Selection should be based on your specific research application, with monoclonals preferred when distinguishing closely related isoforms and polyclonals when detecting potentially modified targets.

How should CK2α antibodies be stored and handled to maintain their activity?

Proper storage and handling are essential for maintaining antibody functionality:

• Storage Temperature: Store at -20°C for lyophilized antibodies; small pack sizes (-SP) are typically supplied either lyophilized or as 0.2 μm filtered solutions in PBS .

• Aliquoting: Upon reconstitution, prepare small single-use aliquots to avoid repeated freeze-thaw cycles that can denature the antibody.

• Working Dilutions: Store working dilutions at 4°C for short-term use (1-2 weeks).

• Buffer Considerations: Prepare antibody dilutions in appropriate buffers with stabilizers; for Western blots, use Immunoblot Buffer Group 1 for optimal results .

• Concentration Information: Maintain records of starting concentration and dilution factors for reproducible experiments.

• Quality Control: Periodically validate stored antibodies against fresh lots, especially for critical experiments.

How can I optimize CK2α antibody conditions for detecting post-translational modifications?

Post-translational modifications (PTMs) of CK2α can significantly affect its activity and interactions. To optimize detection:

• Phospho-Specific Antibodies: For detecting phosphorylated forms (e.g., pS209 on CK2β) , use phospho-specific antibodies with appropriate blocking agents to minimize background.

• Sample Preparation Protocol:

  • Include phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride) in lysis buffers

  • Use gentle detergents to preserve protein-protein interactions

  • Consider short-term treatments with phosphatase inhibitors before cell lysis

• Validation Approaches:

  • Compare samples treated with and without phosphatase

  • Include positive controls with known phosphorylation status

  • Use multiple antibodies targeting different phosphorylation sites

• Optimization Strategies:

  • Test multiple fixation methods for immunocytochemistry

  • Adjust antibody concentration and incubation time

  • Consider enhanced detection systems for low-abundance modifications

• Quantification: Use appropriate software to quantify the ratio of modified to total CK2α for comprehensive analysis.

What are the best strategies for using CK2α antibodies in co-immunoprecipitation experiments?

Co-immunoprecipitation (co-IP) is valuable for studying CK2 protein-protein interactions:

• Antibody Selection: Choose antibodies validated for immunoprecipitation; several clones have been specifically tested for this application .

• Lysis Buffer Optimization:

  • Use mild non-denaturing buffers to preserve protein complexes

  • Include protease inhibitors to prevent degradation

  • Adjust salt concentration based on interaction strength

  • Consider including low concentrations of detergents (0.1-0.5% NP-40 or Triton X-100)

• Pre-clearing Protocol:

  • Pre-clear lysates with control IgG and protein A/G beads

  • This reduces non-specific binding and improves signal-to-noise ratio

• Controls:

  • Include isotype-matched IgG control

  • Use CK2α knockout cells as negative controls

  • Consider reverse co-IP (immunoprecipitate with antibody against interacting protein)

• Detection Method:

  • Use clean detection antibody from different species than IP antibody

  • Consider HRP-conjugated secondary antibodies for reduced background

• Washing Optimization:

  • Determine optimal wash stringency (buffer composition, number of washes)

  • Balance between preserving true interactions and reducing background

How do I troubleshoot inconsistent results when using CK2α antibodies in immunohistochemistry?

Inconsistent immunohistochemistry (IHC) results can be addressed systematically:

• Fixation and Processing:

  • Different fixatives affect epitope accessibility

  • For paraffin-embedded sections, optimize antigen retrieval (tested at 5 μg/mL with overnight incubation at 4°C)

  • Consider section thickness (typically 5-7 μm)

• Antibody Validation:

  • Verify antibody works in your tissue type

  • Test multiple antibody concentrations

  • Include positive control tissues (human brain substantia nigra shows specific nuclear staining in neurons)

• Background Reduction:

  • Block endogenous peroxidase activity

  • Optimize blocking solutions (5-10% normal serum)

  • Consider using specialized detection systems like Anti-Mouse HRP-DAB Cell & Tissue Staining Kit

• Protocol Optimization Table:

• Reproducibility Measures:

  • Standardize all protocol steps

  • Process all comparative samples simultaneously

  • Document exact conditions for each experiment

What are the considerations for using CK2α antibodies in studying signaling pathways?

When investigating CK2α's role in signaling pathways:

• Experimental Design Considerations:

  • Include appropriate time points to capture kinase activation dynamics

  • Consider both nuclear and cytoplasmic fractionation, as CK2α localizes to both compartments

  • Use complementary approaches (Western blotting, immunofluorescence, kinase assays)

• Pathway Activation Monitoring:

  • Monitor CK2α substrates phosphorylation status

  • Consider use of phospho-specific antibodies for CK2α targets

  • Combine with inhibitor studies to confirm pathway specificity

• Context-Dependent Regulation:

  • Account for cell type-specific expression patterns

  • Consider growth conditions that might affect CK2 activity

  • Document cell confluence and passage number

• Interaction Partners:

  • Design experiments to detect CK2α interaction with regulatory β subunits

  • Consider subcellular localization changes upon pathway activation

  • Use appropriate controls when studying protein-protein interactions

• Data Integration Approach:

  • Correlate CK2α activity with downstream effector activation

  • Use systems biology approaches to map pathway interconnections

  • Consider mathematical modeling to understand kinase dynamics

How can I use CK2α antibodies to investigate its role in specific diseases?

CK2α has been implicated in various pathologies, particularly cancer:

• Disease-Specific Tissue Analysis:

  • Compare CK2α expression in normal versus diseased tissues

  • Use immunohistochemistry on tissue microarrays for large-scale studies

  • Quantify expression differences using image analysis software

• Correlation with Clinical Parameters:

  • Design studies to correlate CK2α expression with disease progression

  • Consider subcellular localization changes in disease states

  • Integrate with patient outcome data for prognostic studies

• Cell Line Models:

  • Compare CK2α expression across disease-relevant cell lines

  • Combine with genetic manipulation (siRNA, CRISPR) to assess functional consequences

  • Use HAP1 CK2α knockout cell lines as controls

• Methodological Protocol:

  • Standardize tissue collection and processing

  • Use automated staining platforms when possible

  • Include multiple antibodies to confirm findings

  • Develop scoring systems for CK2α expression levels

• Translational Considerations:

  • Correlate with other molecular markers in the disease

  • Consider potential as diagnostic, prognostic, or therapeutic target

  • Validate findings across independent cohorts

What are the latest techniques for quantifying CK2α expression levels using antibody-based methods?

Advanced quantification methods provide more precise data:

• Simple Western Technology:

  • Automated capillary-based Western blotting system

  • Provides higher reproducibility and quantitative capabilities

  • Successfully applied to detect CK2α at approximately 50-52 kDa in human and mouse cell lines

  • Requires 0.5 mg/mL lysate concentration with 10 μg/mL antibody dilution

• Multiplexed Immunofluorescence:

  • Simultaneously detect CK2α with other proteins

  • Use different fluorophore-conjugated secondary antibodies

  • Quantify co-localization with interaction partners

  • Optimize antibody panels to avoid cross-reactivity

• Image-Based Cytometry:

  • Combine with cell identification markers for heterogeneous populations

  • Use nuclear counterstains like DAPI for localization studies

  • Apply machine learning algorithms for automated analysis

  • Provides single-cell resolution data on protein expression

• Quantitative Considerations Table:

• Internal Controls:

  • Always include housekeeping proteins for normalization

  • Use recombinant protein standards for absolute quantification

  • Consider spike-in controls for process validation

How can I address cross-reactivity issues between CK2α and CK2α' when using antibodies?

Distinguishing between the highly similar CK2α and CK2α' isoforms is challenging:

• Epitope Selection Strategy:

  • Choose antibodies targeting regions with sequence differences between isoforms

  • Verify the exact epitope recognized by commercial antibodies

  • Consider custom antibody development for isoform-specific detection

• Validation Protocol:

  • Use knockout cell lines specific for each isoform

  • Test antibody against recombinant CK2α and CK2α' proteins

  • Include competition assays with purified proteins

• Western Blot Differentiation:

  • Leverage slight molecular weight differences (CK2α: 42-47 kDa)

  • Use high-resolution gel systems (10-12% polyacrylamide)

  • Optimize running conditions for better separation

• Specificity Testing:

  • Compare HAP1 CK2α knockout cells versus wild-type cells

  • Conduct siRNA knockdown specific to each isoform

  • Use immunofluorescence to assess antibody specificity in situ

• Alternative Approaches:

  • Consider mRNA analysis (RT-PCR, RNA-seq) to complement protein studies

  • Use mass spectrometry to identify isoform-specific peptides

  • Employ functional assays that distinguish between isoforms