Phospho-CSNK2A1 (Y255) Antibody

What is the Phospho-CSNK2A1 (Y255) Antibody and what does it detect?

The Phospho-CSNK2A1 (Y255) antibody is a specialized immunological reagent that specifically recognizes and binds to Casein Kinase II alpha (CSNK2A1) only when phosphorylated at tyrosine residue 255. This antibody serves as a valuable tool for detecting the phosphorylated form of CSNK2A1, allowing researchers to study the activation state of this important kinase in various cellular contexts. The antibody is typically produced by immunizing rabbits with synthesized peptides derived from human Casein Kinase II alpha around the phosphorylation site of Tyr255 .

The specificity of this antibody is crucial for distinguishing between the phosphorylated and non-phosphorylated forms of CSNK2A1, enabling researchers to investigate phosphorylation-dependent signaling pathways and regulatory mechanisms. During production, manufacturers often remove antibodies against non-phospho peptides through chromatography using corresponding non-phospho peptides to ensure high specificity .

What are the recommended applications for Phospho-CSNK2A1 (Y255) antibody?

Phospho-CSNK2A1 (Y255) antibody has been validated for multiple experimental applications:

• Western Blotting (WB): Typically used at dilutions between 1:500-1:2000, this application allows detection of phosphorylated CSNK2A1 in cell or tissue lysates, providing information about expression levels and phosphorylation status .

• Immunohistochemistry (IHC): At dilutions of 1:100-1:300, the antibody can detect phosphorylated CSNK2A1 in tissue sections, offering insights into spatial distribution and expression patterns in different cell types or subcellular compartments .

• Enzyme-Linked Immunosorbent Assay (ELISA): Using a dilution of approximately 1:10000, researchers can quantitatively measure phosphorylated CSNK2A1 levels in various samples .

When designing experiments, researchers should perform titration experiments to determine the optimal antibody concentration for their specific experimental conditions, sample types, and detection methods.

What is the biological significance of CSNK2A1 Y255 phosphorylation?

The phosphorylation of CSNK2A1 at tyrosine 255 represents an important regulatory mechanism with significant implications for cellular function. CSNK2A1 (Casein Kinase 2 alpha 1) plays critical roles in numerous cellular processes, with recent research highlighting its importance in primary cilia formation and function .

CSNK2A1 has been identified as a modulator of cilia trafficking and stability, with close functional relationships to TTBK2 (Tau Tubulin Kinase 2). Interestingly, CSNK2A1 and TTBK2 appear to have opposing functions in establishing and maintaining ciliary structure . The phosphorylation state at Y255 may influence these interactions and the regulatory balance between these kinases.

Additionally, mutations in CSNK2A1 are associated with Okur-Chung neurodevelopmental syndrome (OCNDS), characterized by dysmorphic facial features and neurological impairments. Recent studies suggest that these mutations alter cilia morphology, potentially establishing a link between CSNK2A1 ciliary function and the pathophysiology of OCNDS .

How should I optimize Western blot protocols for detecting phosphorylated CSNK2A1?

Optimizing Western blot protocols for phosphorylated proteins requires special considerations:

• Sample Preparation:

  • Harvest cells or tissues in the presence of phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, and phosphatase inhibitor cocktails) to preserve phosphorylation states

  • Perform rapid lysis at cold temperatures to minimize dephosphorylation

  • Consider using cellular fractionation methods like the Minute™ Cytoplasmic and Nuclear Fractionation kit, as CSNK2A1 has been shown to localize to the nuclear fraction in some cell types

• Running Conditions:

  • Use freshly prepared SDS-PAGE gels (10-12%) for optimal resolution

  • Load appropriate positive controls (e.g., HeLa cell lysates have been validated for detection of phospho-CSNK2A1)

• Transfer and Blocking:

  • Perform rapid transfer to PVDF or nitrocellulose membranes

  • Block with 5% BSA in TBST rather than milk, as milk contains phosphoproteins that may interfere with detection

• Antibody Incubation:

  • Start with a 1:1000 dilution of the Phospho-CSNK2A1 (Y255) antibody in 5% BSA/TBST

  • Incubate overnight at 4°C for optimal binding

  • Include appropriate controls, such as parallel blots with non-phospho-specific CSNK2A1 antibody

• Validation Controls:

  • Consider using phosphopeptide competition assays where available to confirm specificity of the signal

  • As demonstrated in published results, compare signals from multiple cell lines (e.g., HeLa and Jurkat cells) to verify consistency

What experimental approaches can be used to study the functional relationship between CSNK2A1 and primary cilia?

Several methodological approaches can be employed to investigate the functional relationship between CSNK2A1 and primary cilia:

• Genetic Manipulation:

  • CRISPR-Cas9 genome editing to generate CSNK2A1 knockout cell lines, as demonstrated in published studies using MEFs

  • Rescue experiments with wild-type and mutant CSNK2A1 constructs to determine structure-function relationships

  • RNAi-mediated knockdown for temporary depletion studies

• Cilia Analysis:

  • Immunofluorescence microscopy using antibodies against ciliary markers (acetylated α-tubulin, ARL13B) and CSNK2A1

  • Quantitative assessment of cilia frequency, length, and morphology

  • Time-lapse imaging to evaluate cilia dynamics and stability

• Functional Assays:

  • Microtubule stability assays using nocodazole treatment to assess ciliary stability in the presence or absence of CSNK2A1

  • Ciliary trafficking assays to monitor the movement of IFT proteins, GLI2, and KIF7

  • Sonic Hedgehog (Shh) pathway activation assays to assess functional consequences of CSNK2A1 manipulation

• Interaction Studies:

  • Co-immunoprecipitation to identify protein-protein interactions

  • Proximity ligation assays to visualize interactions in situ

  • Assessment of the relationship between CSNK2A1 and TTBK2, which have been shown to have opposing functions

These methodologies provide complementary data sets that can collectively reveal the role of CSNK2A1 phosphorylation in ciliary biology and related pathologies.

What are the key considerations for immunohistochemical detection of phospho-CSNK2A1 in tissue samples?

Successful immunohistochemical detection of phosphorylated proteins in tissue samples requires careful attention to several critical factors:

• Tissue Preservation:

  • Rapid fixation is essential to preserve phosphorylation states

  • Phosphatase activity continues post-mortem, so minimize the time between tissue collection and fixation

  • Consider using phosphatase inhibitors during tissue processing

• Antigen Retrieval:

  • Tris-EDTA buffer (pH 9.0) has been validated for antigen retrieval when detecting phospho-CSNK2A1

  • Heat-induced epitope retrieval methods (pressure cooker or microwave) are generally more effective than enzymatic methods for phospho-epitopes

• Protocol Optimization:

  • Antibody dilution: Begin with 1:100-1:300 as recommended

  • Incubation time: Overnight incubation at 4°C often yields optimal results

  • Signal amplification: Consider using polymer-based detection systems for enhanced sensitivity

• Controls:

  • Positive control: Human colon cancer tissue has been validated for phospho-CSNK2A1 detection

  • Negative controls: Include secondary antibody-only controls and, when available, tissues known to lack CSNK2A1 expression

  • Peptide competition: Pre-incubation of the antibody with phospho-peptides should eliminate specific staining

• Dual Staining:

  • Consider dual immunofluorescence or immunohistochemistry to co-localize phospho-CSNK2A1 with other markers

  • For ciliary studies, co-staining with ciliary markers (acetylated α-tubulin, ARL13B) can provide valuable spatial information

By addressing these considerations, researchers can obtain reliable and reproducible immunohistochemical data on phospho-CSNK2A1 localization and expression in tissue samples.

How can I verify the specificity of the phospho-CSNK2A1 antibody signal in my experiments?

Verifying antibody specificity is crucial for generating reliable data. Several approaches can be used to confirm the specificity of phospho-CSNK2A1 (Y255) antibody signals:

• Phosphopeptide Competition Assays:

  • Pre-incubate the antibody with the phosphorylated peptide used as the immunogen

  • In parallel, pre-incubate with the non-phosphorylated version of the same peptide

  • The phosphopeptide should block specific binding while the non-phosphopeptide should not

  • This approach has been validated in Western blot applications, where the phospho-peptide blocked detection in Jurkat cell lysates

• Genetic Approaches:

  • Use CSNK2A1 knockout cells as negative controls

  • Compare wild-type cells to cells expressing CSNK2A1 with a Y255F mutation (preventing phosphorylation)

  • Perform siRNA knockdown experiments to confirm signal reduction correlates with protein depletion

• Phosphatase Treatment:

  • Treat one sample set with lambda phosphatase before analysis

  • The phospho-specific signal should disappear after phosphatase treatment

• Cross-validation with Multiple Techniques:

  • Confirm results using different detection methods (e.g., if using Western blot, validate with ELISA or immunofluorescence)

  • Use antibodies from different sources or clones that recognize the same phosphorylation site

• Phospho-ELISA Validation:

  • Perform comparative ELISA testing using phospho and non-phospho peptides

  • Look for selective detection of the phosphorylated form, as demonstrated in published validation data

These validation approaches provide complementary evidence for antibody specificity and should be included in experimental design whenever possible.

What are the potential causes and solutions for weak or absent phospho-CSNK2A1 signals in Western blots?

Several factors can contribute to weak or absent signals when detecting phosphorylated proteins:

Additionally, when working specifically with phospho-CSNK2A1:

• Consider using nuclear fractionation methods, as CSNK2A1 has been observed in the nuclear fraction of HeLa cells

• Verify experimental conditions that promote or inhibit Y255 phosphorylation

• Check if different cell types show variation in phosphorylation levels

• Remember that phosphorylation is often a dynamic and transient modification, so timing of cell collection is critical

How do I interpret contradictory results between different antibody-based methods when studying phospho-CSNK2A1?

Contradictory results between different antibody-based methods are not uncommon when studying phosphoproteins. A systematic approach to reconciling these contradictions includes:

• Consider Method-Specific Factors:

  • Western blotting denatures proteins, potentially exposing epitopes hidden in native conditions

  • ELISA maintains proteins in more native states, which may affect epitope accessibility

  • IHC involves fixation and antigen retrieval, which can modify epitope presentation

  • Each method may have different sensitivity thresholds and dynamic ranges

• Evaluate Sample Preparation Effects:

  • Different lysis buffers may preserve phosphorylation states to varying degrees

  • Fixation methods for IHC/ICC can affect phospho-epitope preservation

  • Storage conditions and freeze-thaw cycles can lead to differential dephosphorylation

• Analyze Technical Variables:

  • Antibody concentrations should be optimized for each technique (e.g., 1:500-1:2000 for WB vs. 1:100-1:300 for IHC)

  • Detection systems vary in sensitivity (chemiluminescence vs. fluorescence)

  • Blocking reagents may affect antibody binding differently (BSA vs. milk protein)

• Biological Context:

  • Cell type-specific differences in phosphorylation networks

  • Subcellular localization variations (e.g., nuclear enrichment in some contexts)

  • Temporal dynamics of phosphorylation events

• Reconciliation Strategies:

  • Use complementary approaches (e.g., mass spectrometry) to validate phosphorylation

  • Consider kinase/phosphatase assays to directly measure enzymatic activity

  • Employ genetic approaches (Y255F mutation) to confirm specificity

  • Perform time-course experiments to capture dynamic changes

When faced with contradictory results, document all experimental variables and systematically test each factor to identify the source of discrepancy. This process not only resolves contradictions but often leads to new biological insights about the regulation and function of phospho-CSNK2A1.

How can phospho-CSNK2A1 antibodies be used to investigate the relationship between CSNK2A1 and TTBK2 in ciliary regulation?

Research has revealed an intriguing antagonistic relationship between CSNK2A1 and TTBK2 in ciliary regulation, providing fertile ground for advanced investigations:

• Co-localization and Interaction Studies:

  • Dual immunofluorescence staining with phospho-CSNK2A1 and TTBK2 antibodies to examine spatial relationships at basal bodies and within cilia

  • Proximity ligation assays to detect close associations between these proteins in situ

  • Co-immunoprecipitation experiments to assess physical interactions and how these may be altered by phosphorylation states

• Genetic Interaction Analyses:

  • Create single and double knockouts/knockdowns of CSNK2A1 and TTBK2

  • Assess ciliary phenotypes in these genetic backgrounds to quantify suppression or enhancement effects

  • As reported, CSNK2A1 knockout partially rescues ciliary phenotypes in TTBK2 hypomorphic mutant cells, suggesting antagonistic functions

• Phosphorylation Dynamics:

  • Time-course studies using phospho-CSNK2A1 antibodies following ciliary induction

  • Examination of how TTBK2 activity affects CSNK2A1 Y255 phosphorylation and vice versa

  • Correlation of phosphorylation patterns with ciliary assembly and disassembly events

• Downstream Effector Analysis:

  • Investigation of how CSNK2A1 and TTBK2 differentially affect IFT complex recruitment

  • Assessment of actin dynamics at the ciliary base, as CSNK2A1 has been implicated in regulating actin modulators

  • Evaluation of ciliary trafficking defects in various genetic backgrounds using markers for IFT81, GLI2, and KIF7

• Functional Readouts:

  • Sonic Hedgehog pathway activation assays to correlate signaling outcomes with phosphorylation states

  • Ciliary stability tests using nocodazole treatment to compare wild-type, single mutant, and double mutant conditions

  • Live imaging of ciliary dynamics using fluorescently tagged proteins in different genetic backgrounds

These approaches collectively provide a comprehensive framework for dissecting the molecular mechanisms by which CSNK2A1 and TTBK2 antagonistically regulate ciliary structure and function.

What methodologies can be employed to investigate the role of phospho-CSNK2A1 in Okur-Chung neurodevelopmental syndrome (OCNDS)?

Investigating the role of phospho-CSNK2A1 in OCNDS requires integrating clinical, molecular, and cellular approaches:

• Patient-Derived Models:

  • Generate induced pluripotent stem cells (iPSCs) from OCNDS patients

  • Differentiate iPSCs into relevant cell types (neurons, neural progenitors)

  • Use phospho-CSNK2A1 antibodies to assess phosphorylation patterns in patient cells compared to controls

• Mutation-Specific Analysis:

  • Create isogenic cell lines expressing OCNDS-associated CSNK2A1 mutations

  • Evaluate Y255 phosphorylation states in mutant backgrounds

  • Determine if disease mutations affect Y255 phosphorylation directly or indirectly

• Ciliary Morphology and Function:

  • Examine cilia formation, length, and stability in OCNDS models

  • Research has shown that CSNK2A1 mutations alter cilia morphology, potentially linking CSNK2A1 ciliary function to OCNDS

  • Assess ciliary trafficking and signaling pathway activation in disease models

• Phosphoproteomics:

  • Perform global phosphoproteomic analysis of control versus OCNDS cells

  • Identify differentially phosphorylated substrates that might contribute to disease pathology

  • Use phospho-CSNK2A1 antibodies to immunoprecipitate interacting proteins for mass spectrometry analysis

• Animal Models:

  • Generate mouse models harboring OCNDS-associated mutations

  • Analyze developmental phenotypes with particular attention to neurological features

  • Perform tissue-specific immunohistochemistry using phospho-CSNK2A1 antibodies

• Therapeutic Screening:

  • Use phospho-CSNK2A1 levels as a biomarker for screening potential therapeutic compounds

  • Test kinase inhibitors or modulators that might normalize aberrant phosphorylation patterns

  • Evaluate compounds that target downstream effectors in ciliary regulation pathways

This multidisciplinary approach can provide insights into how CSNK2A1 mutations and phosphorylation abnormalities contribute to the pathophysiology of OCNDS, potentially revealing therapeutic targets.

How can quantitative phosphoproteomic approaches complement antibody-based detection of phospho-CSNK2A1?

Quantitative phosphoproteomics offers powerful complementary approaches to antibody-based detection methods:

• Global Phosphorylation Landscape Analysis:

  • Mass spectrometry-based phosphoproteomics can simultaneously measure thousands of phosphorylation sites

  • This approach provides context for Y255 phosphorylation within the broader phosphoproteome

  • Allows identification of co-regulated phosphorylation events that may not be detected by targeted antibody approaches

• Absolute Quantification:

  • Selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) can provide absolute quantification of phospho-CSNK2A1

  • Synthetic phosphopeptides containing the Y255 site can serve as internal standards

  • This approach overcomes the semi-quantitative nature of antibody-based methods

• Stoichiometry Determination:

  • Mass spectrometry can determine the fraction of CSNK2A1 phosphorylated at Y255

  • This information is difficult to obtain using antibody-based methods alone

  • Understanding stoichiometry is crucial for interpreting biological significance

• Multi-site Phosphorylation Analysis:

  • MS/MS fragmentation can identify multiple phosphorylation sites on a single CSNK2A1 molecule

  • This reveals potential cross-talk between Y255 and other phosphorylation sites

  • Helps determine if Y255 phosphorylation is part of a sequential or combinatorial phosphorylation code

• Integration with Antibody-Based Methods:

  • Use phospho-specific antibodies for immunoprecipitation followed by mass spectrometry (phospho-IP-MS)

  • Validate mass spectrometry findings with antibody-based assays in larger sample sets

  • Combine spatial information from immunohistochemistry with quantitative data from mass spectrometry

• Experimental Design Considerations:

  • Include phosphatase inhibitors during sample preparation

  • Consider enrichment strategies for phosphopeptides (TiO2, IMAC, phospho-tyrosine antibodies)

  • Implement appropriate normalization methods for accurate quantification

By integrating antibody-based detection with quantitative phosphoproteomics, researchers can gain comprehensive insights into the regulation and function of CSNK2A1 Y255 phosphorylation in normal and pathological conditions.

What are the emerging research directions involving phospho-CSNK2A1 in ciliopathies and neurodevelopmental disorders?

Several promising research directions are emerging at the intersection of phospho-CSNK2A1 biology, ciliopathies, and neurodevelopmental disorders:

• Mechanistic Understanding of CSNK2A1 in Ciliary Regulation:

  • Further investigation of the antagonistic relationship between CSNK2A1 and TTBK2

  • Elucidation of the precise molecular mechanisms by which CSNK2A1 regulates ciliary trafficking

  • Identification of direct substrates of CSNK2A1 within the ciliary apparatus

• Developmental Neurobiology:

  • Characterization of CSNK2A1 function in neural development and circuitry formation

  • Investigation of how OCNDS-associated mutations affect neuronal ciliogenesis and signaling

  • Exploration of the link between ciliary defects and cognitive/behavioral phenotypes in OCNDS

• Therapeutic Interventions:

  • Development of small molecule modulators that can normalize aberrant CSNK2A1 activity

  • Exploration of gene therapy approaches for severe OCNDS cases

  • Investigation of downstream pathway interventions that might bypass CSNK2A1 dysfunction

• Biomarker Development:

  • Validation of phospho-CSNK2A1 as a diagnostic or prognostic biomarker for ciliopathies

  • Development of high-throughput assays for Y255 phosphorylation in clinical samples

  • Correlation of phosphorylation patterns with disease severity or progression

• Cross-disease Comparisons:

  • Comparative analysis of CSNK2A1 function across different ciliopathies

  • Investigation of potential overlapping mechanisms between OCNDS and other neurodevelopmental disorders

  • Exploration of shared therapeutic targets across related conditions

These emerging directions promise to deepen our understanding of how CSNK2A1 phosphorylation contributes to cellular function in normal development and disease states, potentially leading to novel diagnostic and therapeutic approaches for OCNDS and related disorders.

What are the technical limitations of current phospho-CSNK2A1 antibodies and how might they be overcome in future research?

Current phospho-specific antibodies face several technical limitations that influence experimental outcomes. Understanding these limitations and developing strategies to overcome them will enhance future research:

• Current Limitations:

  • Batch-to-batch variability affecting reproducibility of results

  • Limited dynamic range for quantitative applications

  • Potential cross-reactivity with similar phospho-epitopes

  • Challenges in detecting low abundance phosphorylation events

  • Variability in performance across different applications (WB, IHC, ELISA)

• Emerging Technologies to Overcome Limitations:

  • Development of recombinant monoclonal antibodies for improved consistency

  • Implementation of single-chain variable fragments (scFvs) for enhanced specificity

  • Utilization of synthetic binding proteins (aptamers, affimers) as alternatives to traditional antibodies

  • Application of proximity ligation assays for improved sensitivity and specificity

  • Integration of CRISPR-based tagging strategies to monitor endogenous phosphorylation events

• Complementary Approaches:

  • Combining antibody-based detection with mass spectrometry validation

  • Development of FRET-based biosensors for real-time monitoring of CSNK2A1 phosphorylation

  • Implementation of gene-edited cell lines expressing phosphomimetic or phosphodeficient CSNK2A1 variants

  • Utilization of nanobody technology for improved access to sterically hindered epitopes

• Standardization Efforts:

  • Establishment of reference standards for phospho-CSNK2A1 detection

  • Development of quantitative calibration methods for phospho-specific antibodies

  • Creation of community-wide validation protocols for phospho-specific antibodies