Phospho-CSNK2B (S209) Antibody

What is Phospho-CSNK2B (S209) Antibody and what does it specifically detect?

Phospho-CSNK2B (S209) Antibody is a rabbit polyclonal antibody that specifically recognizes the casein kinase II beta subunit (CSNK2B) when phosphorylated at serine 209. This antibody has been designed to detect endogenous levels of CSNK2B protein only when this specific serine residue is phosphorylated . The antibody is generated using a synthesized peptide derived from human Casein Kinase II beta surrounding the phosphorylation site of S209 as the immunogen . This specificity makes it a valuable tool for studying the phosphorylation state of CSNK2B in various cellular contexts and signaling pathways, particularly in research focused on protein kinase regulation and Wnt signaling .

What species reactivity can I expect with this antibody?

The Phospho-CSNK2B (S209) Antibody demonstrates confirmed reactivity across multiple mammalian species, including human, mouse, and rat samples . This cross-species reactivity has been validated through various techniques including Western blotting, immunohistochemistry, and ELISA applications . The conservation of this phosphorylation site across these species indicates its biological significance and allows for comparative studies across different model organisms. When designing experiments, researchers can confidently use this antibody for studies involving human cell lines as well as common rodent models without concerns about species-specific limitations .

What are the recommended applications and dilutions for this antibody?

The Phospho-CSNK2B (S209) Antibody has been validated for multiple research applications including Western blot (WB), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA) . For optimal results, the following dilution ratios are recommended:

These dilution ratios have been empirically determined to provide the best signal-to-noise ratio for each application . For Western blot applications, the antibody has been successfully tested on various tissue lysates including mouse testis, rat thymus, and human cell lines . When optimizing protocols for your specific experimental conditions, it is advisable to start with these recommended dilutions and adjust as necessary based on signal intensity and background levels.

How should I design control experiments to validate phospho-specificity in my experimental system?

To properly validate the phospho-specificity of the CSNK2B (S209) antibody in your experimental system, multiple controls should be incorporated. First, include a phosphatase treatment control where a portion of your sample is treated with lambda phosphatase to remove phosphate groups . This should result in loss of signal when probed with the phospho-specific antibody while maintaining signal with a total CSNK2B antibody. Second, incorporate kinase inhibition experiments using specific inhibitors of casein kinase II to demonstrate reduced phosphorylation at S209 . Additionally, consider using cell lines with CSNK2B knockdown or knockout as negative controls. For more advanced validation, generating a phospho-null mutant (S209A) through site-directed mutagenesis would provide definitive evidence of antibody specificity . These control experiments should be conducted simultaneously with your primary experiments to ensure the observed signals are genuinely representing phosphorylated CSNK2B at serine 209.

What are the optimal fixation and antigen retrieval methods for detecting phospho-CSNK2B in IHC applications?

For optimal detection of phospho-CSNK2B (S209) in immunohistochemistry applications, proper fixation and antigen retrieval are critical. Phospho-epitopes are particularly sensitive to fixation conditions. For formalin-fixed paraffin-embedded (FFPE) tissues, limit fixation time to 24 hours at room temperature using 10% neutral buffered formalin to preserve phospho-epitopes . For antigen retrieval, heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) has shown superior results compared to EDTA-based buffers for this particular phospho-epitope . The recommended protocol involves heating sections in citrate buffer for 20 minutes at 95-98°C followed by a 20-minute cooling period. For frozen sections, a brief 10-minute fixation in 4% paraformaldehyde is sufficient. Additionally, including phosphatase inhibitors (10 mM sodium fluoride, 1 mM sodium orthovanadate) in all buffers during sample preparation is essential to prevent dephosphorylation of the target epitope . These methods help maintain the integrity of the phosphorylated serine residue while ensuring accessibility for antibody binding.

How can I optimize protein extraction to preserve phosphorylation status of CSNK2B?

Preserving the phosphorylation status of CSNK2B during protein extraction requires special consideration given the labile nature of phosphorylation modifications. The recommended extraction protocol involves using ice-cold RIPA buffer supplemented with a comprehensive phosphatase inhibitor cocktail . Specifically, include 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 1 mM PMSF along with commercial protease inhibitor cocktails . Maintain samples at 4°C throughout the extraction process, and limit the number of freeze-thaw cycles as these can alter phosphorylation status. For tissues, rapid freezing in liquid nitrogen followed by homogenization in the inhibitor-enriched buffer is essential. The extraction should be completed swiftly, ideally within 30 minutes of cell lysis . Additionally, reducing phosphatase activity by adding 10 mM EDTA and adjusting the pH to slightly acidic conditions (pH 6.8-7.0) can further protect phosphorylated residues. Following extraction, samples should be immediately denatured in Laemmli buffer and heated to 95°C for 5 minutes to inactivate any remaining phosphatases before storing at -80°C for subsequent analysis.

How does phosphorylation at S209 regulate CSNK2B function in the context of protein complexes?

Phosphorylation of CSNK2B at serine 209 serves as a critical regulatory mechanism for casein kinase II holoenzyme function and complex formation. CSNK2B exists as part of a tetrameric complex composed of two catalytic subunits (alpha and alpha-prime) and two regulatory beta subunits . The phosphorylation at S209, which resides in the C-terminal regulatory domain, modulates the interaction between CSNK2B and the catalytic subunits, affecting the substrate selectivity and basal activity of the holoenzyme . Research indicates that this phosphorylation event can induce conformational changes that either enhance or restrict access of certain substrates to the catalytic site. Additionally, S209 phosphorylation influences CSNK2B's ability to interact with other cellular proteins beyond the holoenzyme, including components of the Wnt signaling pathway and chromatin-associated factors like PcG protein complexes . To investigate these interactions experimentally, co-immunoprecipitation studies using the phospho-specific antibody compared to a total CSNK2B antibody can reveal phosphorylation-dependent protein interactions. Crosslinking mass spectrometry approaches have further demonstrated that S209 phosphorylation alters the binding partners of CSNK2B in a cell cycle-dependent manner, suggesting temporal regulation of CSNK2B-containing complexes through this phosphorylation event .

What are the technical challenges in quantifying phospho-CSNK2B levels across different subcellular compartments?

Quantifying phospho-CSNK2B (S209) levels across different subcellular compartments presents several technical challenges requiring specialized approaches. CSNK2B distributes across multiple cellular locations including the cytoplasm, nucleus, nucleoplasm, and plasma membrane , with evidence suggesting that phosphorylation status may influence this distribution. The primary challenge is maintaining spatial information while preserving phosphorylation status during sample preparation. Subcellular fractionation protocols must be modified to include phosphatase inhibitors throughout the procedure, and the purity of each fraction must be verified using compartment-specific markers (e.g., GAPDH for cytoplasm, Lamin B1 for nucleus) . Quantitative comparisons between compartments are complicated by the different protein extraction efficiencies from each compartment, necessitating normalization to compartment-specific loading controls rather than total protein . Additionally, standard immunofluorescence approaches for phospho-epitopes may produce misleading results due to epitope masking in certain compartments or in protein complexes. To overcome this, techniques such as Proximity Ligation Assay (PLA) combined with phospho-CSNK2B (S209) antibody can provide more accurate spatial resolution of phosphorylation events . For the most comprehensive analysis, combining biochemical fractionation with quantitative Western blotting and high-resolution imaging approaches like super-resolution microscopy using the phospho-specific antibody will yield the most reliable compartmental distribution data .

How can temporal dynamics of CSNK2B S209 phosphorylation be monitored during cellular signaling events?

Monitoring the temporal dynamics of CSNK2B S209 phosphorylation during cellular signaling events requires sophisticated approaches that combine temporal resolution with phosphorylation specificity. For short-term signaling events (seconds to minutes), researchers should implement rapid cell lysis techniques using pre-warmed lysis buffer containing phosphatase inhibitors followed by quantitative Western blotting with the phospho-CSNK2B (S209) antibody . To capture precise temporal profiles, time-course experiments should include closely spaced time points (e.g., 0, 1, 2, 5, 10, 15, 30, 60 minutes) following stimulus application. For signaling events extending over hours, synchronization of cells (e.g., by serum starvation followed by release) can reduce cell-to-cell variability . For live-cell monitoring, genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) can be developed by fusing CSNK2B with fluorescent proteins and using the phospho-specific antibody fragment as the sensing domain . Additionally, phospho-flow cytometry using the phospho-CSNK2B (S209) antibody allows quantification at the single-cell level, revealing population heterogeneity in phosphorylation responses . For correlating phosphorylation with functional outcomes, parallel samples should be processed for both phosphorylation analysis and downstream functional assays. These approaches collectively provide a comprehensive view of how CSNK2B phosphorylation dynamics correlate with cellular responses to various stimuli, particularly in Wnt signaling or cell cycle progression where CSNK2B plays significant roles .

What are common sources of non-specific signals when using phospho-CSNK2B antibodies, and how can they be mitigated?

Non-specific signals when using phospho-CSNK2B (S209) antibodies can arise from multiple sources and require systematic troubleshooting. The most common source is cross-reactivity with other phosphorylated proteins containing similar epitope sequences. To mitigate this, implement more stringent blocking conditions using 5% BSA rather than milk (which contains phosphoproteins) in TBS-T for at least 2 hours at room temperature . Increasing the wash stringency by adding 0.2% SDS to wash buffers can also reduce non-specific binding. Another frequent issue is detection of dephosphorylated CSNK2B, which can be addressed by verifying phosphatase inhibitor efficacy and freshness . For Western blotting applications specifically, non-specific bands may appear at unexpected molecular weights; running a pre-adsorption control using the immunizing phosphopeptide can confirm which bands represent specific binding . In IHC applications, background staining can result from endogenous peroxidase activity or non-specific antibody binding to tissue components. This can be minimized by thorough peroxidase quenching (3% H₂O₂ for 15 minutes) and using species-specific serum for blocking . Additionally, titrating the antibody to the lowest effective concentration and reducing incubation temperature from room temperature to 4°C can significantly improve signal-to-noise ratio. For definitive validation, always include phosphatase-treated samples and ideally CSNK2B-depleted samples as negative controls .

How can I differentiate between changes in phosphorylation status versus changes in total CSNK2B protein levels?

Differentiating between changes in phosphorylation status versus changes in total CSNK2B protein levels is essential for accurate data interpretation. The recommended methodological approach involves parallel analysis using both the phospho-specific CSNK2B (S209) antibody and a separate antibody that recognizes total CSNK2B regardless of phosphorylation status . When performing Western blots, these should be run on separate aliquots of the same samples under identical conditions, or through membrane stripping and reprobing if protein quantity is limited . The critical analytical step is to calculate the ratio of phospho-CSNK2B to total CSNK2B signal intensity using densitometry software, which normalizes phosphorylation changes to protein abundance . This ratio should be reported rather than phospho-signal alone. For immunofluorescence or IHC studies, dual labeling with both antibodies (using different reporter systems) allows for colocalization analysis and intensity ratio calculations at the cellular level . Additionally, when examining treatments that might affect both phosphorylation and protein stability, include cycloheximide chase experiments to distinguish between these processes . For comprehensive analysis, phospho-specific ELISA assays can provide quantitative data on phospho-to-total ratios across multiple samples simultaneously. Importantly, when reporting results, always present both phospho-specific and total protein data side by side with statistical analysis of the ratio between them to provide transparent evidence of whether observed changes reflect altered phosphorylation events or altered protein expression .

What controls should be included when using phospho-CSNK2B antibody in studies involving kinase inhibitors or phosphatase activators?

When using phospho-CSNK2B (S209) antibody in studies involving kinase inhibitors or phosphatase activators, a comprehensive set of controls is essential for accurate interpretation. First, include a dose-response curve for each inhibitor/activator to establish the optimal concentration that affects CSNK2B phosphorylation without causing cellular toxicity . Second, implement time-course experiments to determine the kinetics of phosphorylation changes, as some compounds may have rapid but transient effects . For specificity controls, utilize structurally distinct inhibitors targeting the same kinase pathway to confirm that observed effects are pathway-specific rather than compound-specific . Additionally, include positive controls where phosphorylation is known to be affected (e.g., treatment with calyculin A as a phosphatase inhibitor should increase phosphorylation signals) and negative controls using inactive analogs of the test compounds .

For advanced validation, complementary genetic approaches should be employed alongside pharmacological treatments: siRNA knockdown of the target kinase should phenocopy the effect of kinase inhibitors, while phosphatase knockdown should resemble phosphatase inhibitor treatment . When examining pathway-specific effects, monitor the phosphorylation status of known upstream and downstream components in the same signaling cascade to establish the position where the inhibitor/activator is exerting its effect . Finally, all phosphorylation data should be normalized to total CSNK2B levels, and loading controls (housekeeping proteins not affected by the treatments) should be included to account for any differences in sample loading . This comprehensive control strategy ensures that any observed changes in CSNK2B phosphorylation can be attributed specifically to the targeted manipulation of kinase or phosphatase activity.

How does phosphorylation at S209 affect CSNK2B's role in Wnt signaling pathway regulation?

Phosphorylation of CSNK2B at serine 209 significantly impacts its regulatory function in the Wnt signaling pathway through multiple molecular mechanisms. CSNK2B serves as a regulatory subunit of casein kinase II holoenzyme, which exists as a tetramer composed of two catalytic subunits (alpha and alpha-prime) and two regulatory beta subunits . S209 phosphorylation modulates CSNK2B's ability to regulate the basal catalytic activity of the alpha subunits, which in turn affects phosphorylation of β-catenin, a central mediator of canonical Wnt signaling . Research indicates that phosphorylated CSNK2B enhances the casein kinase II holoenzyme's ability to phosphorylate β-catenin at residues that promote its stabilization, thereby increasing nuclear accumulation and transcriptional activity . Additionally, phosphorylated CSNK2B exhibits differential binding affinities for other Wnt pathway components, including Dishevelled and Axin, potentially redirecting these scaffold proteins to favor either canonical or non-canonical Wnt signaling branches . To experimentally investigate these relationships, researchers should combine phospho-CSNK2B (S209) antibody detection with TOPFlash reporter assays to correlate CSNK2B phosphorylation status with β-catenin-mediated transcriptional output . Further, co-immunoprecipitation experiments comparing wild-type CSNK2B with phospho-mimetic (S209D/E) and phospho-null (S209A) mutants can reveal how this specific phosphorylation event reorganizes protein interaction networks within the Wnt signaling pathway .

What is the relationship between CSNK2B phosphorylation and its subcellular localization patterns?

The relationship between CSNK2B phosphorylation at S209 and its subcellular localization is complex and context-dependent. CSNK2B exhibits dynamic distribution across multiple cellular compartments including the cytoplasm, nucleus, nucleoplasm, and plasma membrane . Phosphorylation at S209 appears to function as a molecular switch that modulates this distribution pattern. Research utilizing the phospho-CSNK2B (S209) antibody in immunofluorescence studies has revealed that phosphorylated CSNK2B shows enhanced nuclear localization in proliferating cells, suggesting a cell cycle-dependent regulatory mechanism . The molecular basis for this redistribution involves phosphorylation-induced conformational changes that either expose or mask nuclear localization signals within the CSNK2B protein structure . Additionally, phosphorylation at S209 affects CSNK2B's association with membrane proteins and lipid rafts, potentially through altered protein-protein interactions or direct effects on lipid binding domains . To experimentally investigate this relationship, subcellular fractionation followed by Western blotting with both phospho-specific and total CSNK2B antibodies can quantitatively assess compartment-specific distribution patterns . Advanced imaging techniques such as fluorescence recovery after photobleaching (FRAP) using fluorescently tagged CSNK2B variants (wild-type, S209A, and S209D/E) can further illuminate how phosphorylation affects protein mobility and compartmental residence time . Understanding this phosphorylation-dependent localization is particularly relevant when investigating CSNK2B's participation in nuclear processes such as transcriptional regulation and chromatin remodeling versus cytoplasmic signaling events .

How can phospho-CSNK2B antibodies be used to investigate cross-talk between casein kinase II signaling and other major cellular pathways?

Phospho-CSNK2B (S209) antibodies provide a powerful tool for investigating cross-talk between casein kinase II signaling and other major cellular pathways through multiple experimental approaches. For studying pathway intersection points, dual stimulation experiments should be designed where cells are treated with activators of different pathways (e.g., Wnt ligands, growth factors, stress inducers) in various sequences and combinations, followed by quantitative analysis of S209 phosphorylation levels . This reveals whether certain pathways enhance or suppress CSNK2B phosphorylation. To identify upstream kinases responsible for S209 phosphorylation in different contexts, kinase inhibitor screens combined with phospho-specific detection can map the signaling networks converging on CSNK2B . For downstream effects, comparative phosphoproteomic analysis between wild-type cells and those expressing phospho-null (S209A) CSNK2B mutants can reveal how this phosphorylation event propagates signals to other pathways .

The phospho-CSNK2B antibody can also be implemented in proximity-dependent biotinylation (BioID) approaches, where phosphorylated versus non-phosphorylated CSNK2B-BirA* fusion proteins are used to identify differential protein interaction networks . To investigate temporal aspects of cross-talk, synchronized cell populations can be analyzed at different cell cycle stages to determine how CSNK2B phosphorylation coordinates with cell cycle regulators . Additionally, stress response studies examining how oxidative stress, DNA damage, or metabolic perturbations affect CSNK2B phosphorylation can reveal integration with cellular homeostasis pathways . For in vivo relevance, tissue microarrays probed with the phospho-CSNK2B antibody can identify pathological contexts where altered phosphorylation correlates with dysregulation of multiple signaling pathways . These comprehensive approaches collectively illuminate how CSNK2B phosphorylation serves as a node for information integration across diverse cellular signaling networks .

How can phospho-CSNK2B (S209) antibody be utilized in ChIP-seq experiments to study chromatin associations?

Utilizing phospho-CSNK2B (S209) antibody in ChIP-seq experiments requires specialized protocols to effectively capture chromatin associations while preserving phospho-epitopes. CSNK2B associates with chromatin and participates in PcG protein complexes, making this application particularly valuable for understanding its role in transcriptional regulation . The optimal ChIP-seq protocol involves dual crosslinking: first with protein-protein crosslinker DSG (2 mM, 45 minutes) followed by standard formaldehyde treatment (1%, 10 minutes) to effectively capture both direct and indirect DNA interactions . Critical modifications to standard ChIP protocols include supplementing all buffers with phosphatase inhibitors (50 mM NaF, 10 mM Na3VO4) and performing chromatin fragmentation by sonication at 4°C with reduced power settings to preserve phospho-epitopes . For immunoprecipitation, pre-clearing chromatin with protein A/G beads coupled to non-immune IgG helps reduce background, followed by overnight incubation with phospho-CSNK2B (S209) antibody at a 1:50 dilution .

To validate specificity, parallel ChIP experiments should be performed with total CSNK2B antibody and phospho-null mutant (S209A) expressing cells . For data analysis, bioinformatic comparison of phospho-CSNK2B binding sites with datasets for transcription factors, histone modifications, and chromatin remodelers can reveal functional associations . Additionally, sequential ChIP (re-ChIP) using first the phospho-CSNK2B antibody followed by antibodies against suspected interacting partners can confirm co-occupancy at specific genomic loci . This approach has successfully revealed phosphorylation-dependent recruitment of CSNK2B to promoters of genes involved in cell cycle regulation and Wnt signaling, demonstrating how S209 phosphorylation may directly influence transcriptional outcomes .

What are the considerations for using phospho-CSNK2B antibodies in multiplex immunofluorescence studies?

Multiplex immunofluorescence studies using phospho-CSNK2B (S209) antibody require careful optimization to achieve specific signal detection alongside other markers. Since the antibody is rabbit-derived, planning antibody combinations from different host species (mouse, goat, chicken) for other targets minimizes cross-reactivity issues . For sequential staining protocols, the phospho-CSNK2B antibody should be applied first in the sequence at a 1:100 dilution with overnight incubation at 4°C to maximize sensitivity for this potentially low-abundance phospho-epitope . When using tyramide signal amplification (TSA) systems, reduce H2O2 concentration to 0.003% and TSA reagent to 1:150 dilution to prevent overstaining while maintaining phospho-specificity .

Particular attention must be paid to antigen retrieval methods when combining with other antibodies; while citrate buffer (pH 6.0) works optimally for phospho-CSNK2B, some co-markers may require different pH conditions, necessitating compromise or sequential retrieval steps . For spectral separation, assign phospho-CSNK2B to fluorophores in the far-red spectrum (e.g., Alexa Fluor 647) which typically show less autofluorescence background in tissue sections . To validate multiplexed signals, single-stained controls, fluorescence minus one (FMO) controls, and phosphatase-treated sections should be included . For quantitative analysis, nuclear/cytoplasmic ratios of phospho-CSNK2B can be calculated using nuclear counterstains like DAPI as reference, while colocalization with other markers should be analyzed using appropriate coefficients (Pearson's or Mander's) and intensity correlation analyses . These optimized protocols enable simultaneous visualization of phospho-CSNK2B alongside pathway components or cell-type-specific markers, providing spatial context to phosphorylation events within heterogeneous tissues or complex cellular structures .

How can phospho-CSNK2B antibodies be implemented in high-throughput screening approaches?

Implementing phospho-CSNK2B (S209) antibodies in high-throughput screening approaches requires optimization for miniaturized formats while maintaining sensitivity and specificity. For cell-based screens, researchers should develop a robust in-cell ELISA (ICE) protocol in 384-well format, where cells are fixed directly in plates and probed with phospho-CSNK2B antibody at 1:1000 dilution followed by HRP-conjugated secondary antibody . Signal normalization to total CSNK2B (detected in parallel wells) and cell number (via DNA stains) is essential for accurate quantification across large sample sets . Alternatively, homogeneous time-resolved fluorescence (HTRF) assays can be optimized using donor-labeled anti-phospho-CSNK2B (S209) antibody paired with acceptor-labeled anti-CSNK2B antibody, allowing ratiometric detection of phosphorylation status in lysates without washing steps .

For automated microscopy screens (high-content analysis), cells should be seeded at 3000-5000 cells/well in 384-well imaging plates, with immunofluorescence protocols adapted to include shorter incubation times (primary antibody at 1:200 for 2 hours) and automated liquid handling . Image analysis algorithms should be trained to quantify phospho-CSNK2B nuclear/cytoplasmic ratio and colocalization with relevant markers across thousands of individual cells . For biochemical screens targeting modulators of CSNK2B phosphorylation, a phospho-specific ELISA can be developed using immobilized capture antibody against total CSNK2B and detection with the phospho-CSNK2B (S209) antibody . Quality control metrics for HTS implementation should include Z-factor calculation using positive controls (calyculin A treatment) and negative controls (lambda phosphatase treatment), with Z' > 0.5 indicating suitability for screening . These approaches enable screening of chemical libraries, siRNA/CRISPR libraries, or cDNA expression libraries to identify novel regulators of CSNK2B phosphorylation with potential therapeutic relevance in pathways where CSNK2B plays critical roles .