Phospho-CSNK1A1/CSNK1A1L (Y294) Antibody

What is the biological significance of CSNK1A1 Y294 phosphorylation?

The phosphorylation at tyrosine 294 of CSNK1A1 represents a regulatory modification that may influence the kinase's activity and interaction with substrate proteins. CSNK1A1 is primarily known as a serine/threonine kinase that phosphorylates numerous substrates including CTNNB1 (β-catenin) at Ser-45, which is crucial for the negative regulation of canonical Wnt signaling pathway . The Y294 phosphorylation site is located within the amino acid range 260-309 of the protein, potentially affecting its catalytic function or protein-protein interactions . This phosphorylation may modulate CSNK1A1's roles in various cellular processes including cell division, nuclear localization, and membrane transport functions, though more research is needed to fully characterize its specific effects .

What are the recommended applications for Phospho-CSNK1A1 (Y294) antibodies?

Phospho-CSNK1A1 (Y294) antibodies are validated for multiple experimental applications. Based on validation studies, these antibodies are suitable for Western blotting (WB), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA) . These applications allow researchers to detect and quantify the presence of phosphorylated CSNK1A1 at Y294 in various sample types. The antibodies undergo rigorous validation processes including testing on known positive and negative controls to ensure specificity and high affinity . For optimal results, researchers should follow recommended dilution protocols for each specific application and validate the antibody in their specific experimental system.

What are the optimal storage conditions for maintaining Phospho-CSNK1A1 (Y294) antibody activity?

To preserve antibody functionality, Phospho-CSNK1A1 (Y294) antibodies should be stored at -20°C for long-term storage (up to one year) . For frequent use over shorter periods (up to one month), storage at 4°C is recommended . The antibody is typically supplied in a stabilizing solution containing PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide to maintain integrity . Researchers should avoid repeated freeze-thaw cycles as these can significantly diminish antibody activity and specificity. Aliquoting the antibody upon receipt is a recommended practice to minimize freeze-thaw cycles while ensuring long-term stability for critical experiments.

How can I distinguish between CSNK1A1 and CSNK1A1L phosphorylation in experimental systems?

Distinguishing between phosphorylated CSNK1A1 and its paralog CSNK1A1L requires careful experimental design due to their sequence similarity. A dual-approach strategy is recommended: first, use specific antibodies validated against both proteins, then confirm with molecular techniques. For immunoblotting, perform careful molecular weight analysis (CSNK1A1 has a calculated molecular weight of 38.915 kDa) and include appropriate controls. Consider using recombinant proteins as standards to establish clear migration patterns. For definitive differentiation, complement antibody-based methods with mass spectrometry to identify phosphopeptides unique to each protein. RNA interference or CRISPR-based knockdowns targeting either gene can also help validate the specificity of detected signals in your experimental system.

What is the relationship between Y294 phosphorylation and CSNK1A1's role in the beta-catenin destruction complex?

The Y294 phosphorylation of CSNK1A1 may modulate its critical function within the beta-catenin destruction complex, though this relationship requires further investigation. CSNK1A1 is a key component of this complex and phosphorylates β-catenin at Ser-45, which primes it for subsequent phosphorylation by GSK3β and ultimate proteasomal degradation . This process is central to the negative regulation of canonical Wnt signaling. To experimentally assess whether Y294 phosphorylation affects this function, researchers could employ phosphomimetic (Y294E or Y294D) and phospho-dead (Y294F) CSNK1A1 mutants to examine changes in β-catenin phosphorylation efficiency, destruction complex formation, and downstream Wnt target gene expression. Co-immunoprecipitation assays comparing wild-type and mutant CSNK1A1 can reveal potential changes in interaction with other destruction complex components like Axin and APC.

How does CSNK1A1 Y294 phosphorylation status change across different cellular contexts and disease states?

The phosphorylation status of CSNK1A1 at Y294 likely varies across cellular contexts and may be altered in disease states, though comprehensive characterization is still emerging. CSNK1A1 has been implicated as a biomarker in conditions such as Alzheimer's disease and inclusion body myositis . To profile Y294 phosphorylation across contexts, researchers should consider using phospho-specific antibodies in immunohistochemistry of tissue microarrays representing different tissues and disease states. Quantitative phosphoproteomics can provide an unbiased assessment of phosphorylation changes across cell types or in response to stimuli. For disease relevance, analysis of publicly available phosphoproteomic datasets from patient samples can identify potential correlations between Y294 phosphorylation and disease progression. Temporal dynamics should also be considered, as phosphorylation may change during cell cycle progression or in response to specific signaling events.

What kinases and phosphatases regulate the Y294 phosphorylation state of CSNK1A1?

The specific kinases and phosphatases that directly regulate CSNK1A1 Y294 phosphorylation remain to be fully characterized. To identify these regulatory enzymes, researchers could employ several approaches: kinase prediction algorithms to identify potential kinases based on the sequence surrounding Y294; in vitro kinase assays with purified CSNK1A1 and candidate kinases; phosphatase inhibitor screens to observe effects on Y294 phosphorylation levels; and proximity labeling techniques (BioID or APEX) with CSNK1A1 as bait to identify proximal kinases and phosphatases. Given CSNK1A1's roles in multiple signaling pathways including Wnt, mTOR, and inflammasome regulation , its phosphorylation may be influenced by pathway-specific kinases. Time-course experiments following pathway stimulation or inhibition could help establish the regulatory network controlling this modification.

What are the optimal sample preparation techniques for detecting phospho-Y294 CSNK1A1 in different experimental systems?

When designing experiments to detect phospho-Y294 CSNK1A1, rapid sample processing is critical to preserve phosphorylation states. Samples should be processed quickly and maintained at cold temperatures throughout lysis and extraction procedures. A comprehensive phosphatase inhibitor cocktail is essential, particularly including tyrosine phosphatase inhibitors like sodium orthovanadate . For immunoprecipitation experiments, gentler lysis conditions may better preserve protein complexes containing CSNK1A1. When analyzing tissues, consider that CSNK1A1 localizes to multiple cellular compartments including cytoplasm, centrosome, kinetochore, and nuclear speckles , so subcellular fractionation may provide more detailed insights into compartment-specific phosphorylation.

How can I validate the specificity of phospho-Y294 signal in my experimental system?

Establishing the specificity of phospho-Y294 detection requires multiple validation approaches. First, implement peptide competition assays using the phosphopeptide that was used as the immunogen (amino acids 260-309 surrounding Y294) - the phospho-specific signal should be blocked by the phosphopeptide but not by the unphosphorylated version. Second, use CSNK1A1 knockout cell lines as negative controls to confirm antibody specificity . Third, employ phosphatase treatment of half your sample to demonstrate that the signal is phosphorylation-dependent. For genetic validation, express phospho-dead (Y294F) CSNK1A1 mutants in a CSNK1A1-depleted background - these should not be detected by the phospho-specific antibody. Finally, use orthogonal techniques such as Phos-tag gels to separate phosphorylated from non-phosphorylated forms based on mobility shift, rather than relying solely on antibody detection.

What technical considerations should be addressed when designing experiments to study the functional consequences of Y294 phosphorylation?

To study functional consequences of Y294 phosphorylation, consider experimental timing carefully - CSNK1A1 functions change throughout the cell cycle, particularly during mitosis when it localizes to kinetochore fibers . Synchronization protocols may be necessary to capture phase-specific effects. Since CSNK1A1 participates in multiple pathways including Wnt signaling, mTORC1/2 regulation, and inflammasome assembly , design readouts to capture these diverse functions. For Wnt signaling, measure β-catenin phosphorylation and stability along with TCF/LEF reporter activity. For mTOR pathway analysis, examine phosphorylation of downstream targets like S6K and 4EBP1. Consider using proximity labeling techniques to identify phosphorylation-dependent interaction partners under physiological conditions.

What are the key considerations for quantifying phospho-Y294 CSNK1A1 levels in clinical samples?

When analyzing clinical samples for phospho-Y294 CSNK1A1, standardization is essential for reliable quantification. First, establish a standard operating procedure for sample collection and processing, minimizing time to preservation to prevent phosphorylation changes. Second, include reference standards in each experimental batch - consider using recombinant phosphorylated CSNK1A1 protein at known concentrations. For immunohistochemistry applications, use a tissue microarray containing both positive and negative controls to ensure consistent staining across specimens . When developing quantification methods, consider the heterogeneity of clinical samples and establish appropriate normalization strategies - options include normalization to total CSNK1A1, to housekeeping proteins, or using ratio-metric approaches comparing phosphorylated to total protein. Since CSNK1A1 is a potential biomarker for conditions like Alzheimer's disease , validate any quantification method against established clinical metrics.

What are common pitfalls when detecting phospho-Y294 CSNK1A1 and how can they be addressed?

When troubleshooting phospho-Y294 CSNK1A1 detection, remember that phosphorylation states can be extremely labile. If signal is weak despite proper sample preparation, consider enriching for phosphoproteins using titanium dioxide or immunoprecipitation before analysis . For Western blotting applications, optimize transfer conditions for proteins in the 38-39 kDa range where CSNK1A1 migrates . If background remains problematic in immunostaining applications, consider using more stringent washing buffers or adding low concentrations of detergents to reduce non-specific binding. For highly sensitive applications, consider using signal amplification methods like tyramide signal amplification, but validate that these do not introduce artifacts.

How can I optimize the detection of phospho-Y294 CSNK1A1 in low-abundance samples?

For detecting phospho-Y294 CSNK1A1 in samples with low abundance, enrichment strategies are essential. Implement immunoprecipitation with total CSNK1A1 antibodies before probing with phospho-specific antibodies to concentrate the target protein. Consider using phosphotyrosine-specific antibodies for initial enrichment followed by CSNK1A1-specific detection. For enhanced sensitivity in Western blotting, use high-sensitivity chemiluminescent substrates or switch to fluorescent secondary antibodies with digital imaging systems that offer greater dynamic range. In mass spectrometry applications, employ targeted approaches like selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) focused specifically on the Y294-containing peptide. For immunohistochemistry of tissues with low expression, use signal amplification methods like avidin-biotin complexes or polymer-based detection systems . In all cases, extend exposure times but include appropriate negative controls to distinguish specific signal from background.

What strategies can help distinguish between different phosphorylated forms of CSNK1A1 in complex samples?

CSNK1A1 can be phosphorylated at multiple sites, making the specific detection of Y294 phosphorylation challenging in complex samples. To address this, employ multiple separation techniques: use Phos-tag acrylamide gels to separate proteins based on their phosphorylation status; perform 2D gel electrophoresis separating first by isoelectric point (affected by phosphorylation) and then by molecular weight; or use isoelectric focusing to resolve differently phosphorylated forms. For immunological approaches, perform sequential immunoprecipitations – first with phosphotyrosine antibodies, then with CSNK1A1 antibodies. In mass spectrometry analysis, search for multiply phosphorylated peptides and establish their abundance relative to singly phosphorylated forms. When examining CSNK1A1's functions in specific pathways, use contextual stimuli that activate particular pathways (like Wnt activators or mTOR stimulation ) to enrich for pathway-specific phosphorylation patterns.

How can I verify that my observed phenotypes are specifically due to Y294 phosphorylation rather than other CSNK1A1 functions?

Establishing causality between Y294 phosphorylation and observed phenotypes requires careful experimental design. First, implement a genetic complementation approach: deplete endogenous CSNK1A1 using CRISPR or RNAi, then reintroduce either wild-type, phospho-mimetic (Y294E), or phospho-dead (Y294F) versions at physiological levels. Compare phenotypes across these conditions to isolate phosphorylation-specific effects. Second, develop temporal control systems such as optogenetic or chemical-inducible methods to modulate Y294 phosphorylation rapidly and observe immediate consequences. Third, identify and manipulate the specific kinase responsible for Y294 phosphorylation rather than CSNK1A1 itself. Fourth, use domain-specific CSNK1A1 mutants that maintain catalytic activity but alter specific functions to distinguish between kinase-dependent and structural roles. Finally, attempt to identify direct substrates or interactors that specifically recognize the phospho-Y294 form using BioID or phospho-specific protein arrays, then directly manipulate these downstream components to determine if they recapitulate the observed phenotypes.

What are emerging techniques that might enhance our understanding of CSNK1A1 Y294 phosphorylation in disease contexts?

Emerging technologies promise to revolutionize our understanding of CSNK1A1 Y294 phosphorylation dynamics in disease. Spatial proteomics techniques like imaging mass cytometry can map phospho-Y294 CSNK1A1 distribution within heterogeneous tissues at subcellular resolution, particularly relevant for diseases with regional pathology like Alzheimer's where CSNK1A1 serves as a biomarker . Single-cell phosphoproteomics can reveal cell-to-cell variation in phosphorylation status within complex tissues or tumors. For mechanistic studies, genome-wide CRISPR screens with phospho-Y294 levels as readout could identify novel regulators. Proximity labeling methods (TurboID, APEX) with phospho-specific binding domains can identify proteins that selectively interact with phosphorylated CSNK1A1. In animal models, phospho-specific intrabodies or nanobodies could enable real-time tracking of Y294 phosphorylation in living tissues. These approaches collectively hold potential to uncover how this specific modification contributes to CSNK1A1's roles in the Wnt pathway, mTOR signaling, and inflammasome regulation across different disease contexts.