Phospho-CSNK1A1/CSNK1A1L (Tyr294) Antibody

Fundamental Characteristics of Phospho-CSNK1A1/CSNK1A1L (Tyr294) Antibody

Phospho-CSNK1A1/CSNK1A1L (Tyr294) Antibody is a polyclonal antibody specifically designed to detect the casein kinase 1 alpha protein only when phosphorylated at tyrosine 294. This antibody is developed through immunization of rabbits with synthetic phosphopeptide sequences derived from the region surrounding the tyrosine 294 residue of CK1-alpha . The immunogen specifically consists of peptide sequences containing the phosphorylation motif Y-D-Y(p)-T-F, which corresponds to the phosphotyrosine 294 region of the casein kinase 1 alpha protein . The high specificity of this antibody allows researchers to distinguish between phosphorylated and non-phosphorylated forms of the protein, making it valuable for studying post-translational modifications and associated signaling pathways.

The antibody is typically supplied in liquid form, containing 100μL of purified antibody in phosphate buffered saline without magnesium and calcium ions, at pH 7.4, containing 150mM sodium chloride, 0.02% sodium azide, and 50% glycerol . This formulation ensures stability during storage and optimal performance in various experimental applications. The purification process involves affinity chromatography using epitope-specific phosphopeptides, with additional chromatographic steps to remove non-phospho-specific antibodies, enhancing the specificity of the final product .

Target Protein: CSNK1A1/CSNK1A1L Structure and Function

CSNK1A1/CSNK1A1L (Casein Kinase 1 Alpha 1/Casein Kinase 1 Alpha 1 Like) belongs to the casein kinase I family of serine/threonine kinases, which are characterized by their preferential phosphorylation of acidic protein substrates, particularly caseins . These kinases are monomeric proteins with molecular weights ranging from 25 to 55 kDa and are ubiquitously distributed in eukaryotic cells across the nuclei, cytoplasm, and membrane fractions . The CSNK1A1 gene is located on chromosome 5q32 in humans and encodes a protein of approximately 39 kDa .

Casein kinase 1 alpha functions as a critical regulator in multiple cellular processes. It dynamically associates with the CBM (CARMA1-BCL10-MALT1) complex upon T cell receptor engagement, participating in cytokine production and lymphocyte proliferation . Interestingly, it exhibits a dual role by subsequently promoting the phosphorylation and inactivation of CARMA1, thereby providing negative feedback regulation . The protein governs antigen-receptor-induced NF-kappa-B activation and plays a crucial role in human lymphoma cell survival .

One of the most well-documented functions of CSNK1A1 is its participation in the Wnt signaling pathway, where it phosphorylates beta-catenin (CTNNB1) at Serine-45 . This phosphorylation marks beta-catenin for subsequent phosphorylation by GSK-3β and eventual proteasomal degradation, thus negatively regulating Wnt signaling. Additionally, CSNK1A1 may phosphorylate circadian rhythm proteins PER1 and PER2, suggesting its involvement in circadian clock regulation . The protein may also play roles in chromosome segregation during mitosis and cytoskeleton disassembly, which can regulate epithelial cell migration .

Phosphorylation at Tyrosine 294: Significance and Regulation

The phosphorylation of CSNK1A1/CSNK1A1L at tyrosine 294 represents a specific post-translational modification that may alter the protein's function, localization, or interactions. While the search results provide limited information specifically about the functional consequences of Tyr294 phosphorylation, the development of antibodies targeting this site indicates its research significance .

The tyrosine 294 residue is located within a functionally important region of the protein (approximately residues 260-340), suggesting that phosphorylation at this site may influence kinase activity or substrate recognition . The sequence context surrounding Tyr294 (Y-D-Y-T-F) appears to be conserved across species, including humans, mice, and rats, as evidenced by the cross-reactivity of the antibody .

Phosphorylation at tyrosine residues is typically mediated by tyrosine kinases, which are activated in response to various cellular stimuli, including growth factors, cytokines, and cellular stress. The specific kinases responsible for phosphorylating CSNK1A1 at Tyr294 have not been explicitly identified in the search results, representing a potential area for future research.

Experimental Applications and Methodologies

The Phospho-CSNK1A1/CSNK1A1L (Tyr294) Antibody has been validated for multiple experimental applications, making it a versatile tool for research. These applications include Enzyme-Linked Immunosorbent Assay (ELISA), Western Blotting (WB), and Immunohistochemistry (IHC) .

ELISA Applications

For ELISA applications, the antibody has demonstrated high sensitivity with recommended dilutions of 1:5000 . This high dilution factor suggests excellent affinity for the phosphorylated epitope, allowing for sensitive detection of phospho-CSNK1A1 in complex biological samples. ELISA-based detection is particularly useful for quantitative analysis of phosphorylation levels across different experimental conditions.

Western Blotting

In Western Blotting applications, the recommended dilution range is 1:500-1:1000 . The antibody can detect the endogenous levels of CSNK1A1 protein specifically when it is phosphorylated at Tyr294. Western blotting provides information about protein expression levels, molecular weight (approximately 39 kDa for CSNK1A1), and the relative abundance of the phosphorylated form under different experimental conditions.

Immunohistochemistry

For IHC applications, the recommended dilution range is 1:50-1:100 . This application allows for the visualization of phosphorylated CSNK1A1 in tissue sections, providing insights into the spatial distribution of the phosphoprotein in different cell types and subcellular compartments. The relatively low dilution factor for IHC suggests that optimization may be necessary for specific tissue types or fixation methods.

Comparative Analysis with Other CSNK1A1 Antibodies

The Phospho-CSNK1A1/CSNK1A1L (Tyr294) Antibody is one of several phospho-specific antibodies targeting different phosphorylation sites on CSNK1A1. Another notable example is the CSNK1A1 Antibody (Phospho-Tyr321), which recognizes a different phosphorylation site on the same protein .

Both antibodies share similar characteristics in terms of host species, applications, and general formulation, but they target distinct phosphorylation sites on CSNK1A1. The existence of antibodies targeting different phosphorylation sites suggests that CSNK1A1 undergoes multiple phosphorylation events, potentially serving distinct regulatory functions. Researchers may select the appropriate phospho-specific antibody based on their specific research questions and the signaling pathways under investigation.

Research Implications and Future Directions

The Phospho-CSNK1A1/CSNK1A1L (Tyr294) Antibody serves as a valuable tool for investigating several important biological processes. Given the role of CSNK1A1 in Wnt signaling, antibodies detecting specific phosphorylation sites may help elucidate the regulatory mechanisms of this pathway in both normal development and disease states, particularly in cancer where Wnt signaling is frequently dysregulated .

The involvement of CSNK1A1 in T cell receptor signaling and NF-kappa-B activation suggests potential applications in immunological research, including studies of lymphocyte activation, cytokine production, and inflammatory responses . The phospho-specific antibody could help track the activation status of CSNK1A1 during immune cell stimulation and identify novel regulatory mechanisms.

Additionally, the potential role of CSNK1A1 in chromosome segregation during mitosis indicates relevance to cell cycle research and possibly cancer biology . Phosphorylation at Tyr294 might influence these processes, and the antibody provides a means to investigate this relationship.

Future research directions may include:

1. Identifying the kinases and phosphatases that regulate Tyr294 phosphorylation

2. Determining the functional consequences of this phosphorylation on CSNK1A1 activity and substrate specificity

3. Investigating the role of Tyr294 phosphorylation in disease contexts, including cancer and inflammatory disorders

4. Exploring potential cross-talk between different phosphorylation sites on CSNK1A1