Phospho-CTNNB1 (Ser37) Antibody

What does the Phospho-CTNNB1 (Ser37) antibody specifically recognize?

The Phospho-CTNNB1 (Ser37) antibody specifically recognizes β-catenin that has been phosphorylated at the serine 37 position. It detects endogenous levels of Catenin-Beta protein only when phosphorylated at this specific residue (S37) . Depending on experimental conditions and the elapsed time following the phosphorylation event, the antibody can detect both the full-length phospho-β-catenin (Ser37) and degraded/fragmented forms that result immediately after phosphorylation . This specificity makes it valuable for studying the phosphorylation-dependent regulation of β-catenin in the Wnt signaling pathway.

What is the significance of β-catenin phosphorylation at Ser37 in cellular signaling?

Phosphorylation of β-catenin at Ser37 is a critical regulatory event in the canonical Wnt signaling pathway. In the absence of Wnt ligands, β-catenin forms a complex with AXIN1, AXIN2, APC, CSNK1A1, and GSK3B that promotes phosphorylation on N-terminal Ser and Thr residues . Specifically, phosphorylation by GSK3B requires prior phosphorylation of Ser-45 by another kinase, after which phosphorylation proceeds from Thr-41 to Ser-37 and Ser-33 . This sequential phosphorylation triggers ubiquitination by the SCF(BTRC) E3 ligase complex, leading to proteasomal degradation of β-catenin . Therefore, detection of phosphorylation at Ser37 is indicative of β-catenin destined for degradation in the absence of Wnt signaling.

What are the recommended applications for Phospho-CTNNB1 (Ser37) antibodies?

Phospho-CTNNB1 (Ser37) antibodies are suitable for multiple research applications including:

• Western Blot (WB) at dilutions of 1:500-1:2000

• Immunohistochemistry (IHC) at dilutions of 1:100-1:300

• Immunofluorescence (IF) at dilutions of 1:200-1:1000

• ELISA at dilutions of 1:40000

These applications allow researchers to detect and quantify phosphorylated β-catenin in various experimental contexts, from protein expression levels in cell lysates to spatial localization in tissue sections. The specific dilution ranges provided serve as starting points for optimization in individual experimental settings.

What species reactivity can be expected with these antibodies?

According to the product information, Phospho-CTNNB1 (Ser37) antibodies typically show reactivity with human, mouse, and rat samples . Some antibodies, such as those from Merck Millipore, are predicted to cross-react with additional species including xenopus and chicken based on sequence homology . This cross-reactivity is due to the high conservation of the β-catenin sequence around the Ser37 phosphorylation site across different species, making these antibodies versatile tools for comparative studies across model organisms.

How should samples be prepared to optimally detect phosphorylated β-catenin at Ser37?

For optimal detection of phosphorylated β-catenin at Ser37, samples should be prepared with consideration of the rapid degradation of phosphorylated β-catenin. After phosphorylation, β-catenin is rapidly degraded into various fragments over time . Therefore:

• Use phosphatase inhibitors during sample preparation to preserve phosphorylation status

• Process samples quickly and maintain cold temperatures throughout

• Consider the timing of sample collection after stimulation or inhibition of relevant pathways

• For western blotting, be prepared to detect both full-length phospho-β-catenin and degraded fragments

• Include appropriate positive controls (e.g., cells treated with GSK3β activators) and negative controls (e.g., cells treated with Wnt agonists like CHIR99021)

For cell culture experiments examining Wnt signaling, researchers can follow protocols similar to those used in FCS measurements where cells are treated with specific modulators like CHIR99021 (10 μM for 16-26 hours) or LGK974 (5 μM for 5 days) with subsequent Wnt3a stimulation (100 ng/ml) .

What are the key controls that should be included when using Phospho-CTNNB1 (Ser37) antibodies?

When designing experiments with Phospho-CTNNB1 (Ser37) antibodies, several controls should be included:

• Positive controls: Cells or tissues with known high levels of β-catenin phosphorylation at Ser37 (e.g., cells without Wnt stimulation where the destruction complex is active)

• Negative controls:

  • Cells treated with GSK3β inhibitors like CHIR99021, which prevent phosphorylation at Ser37

  • Cells expressing β-catenin with S37A mutation that cannot be phosphorylated at this position

• Specificity controls:

  • Peptide competition assays using the phosphopeptide used as the immunogen

  • Use of total β-catenin antibody in parallel to determine the ratio of phosphorylated to total protein

• Technical controls:

  • Secondary antibody-only controls to assess non-specific binding

  • Loading controls (e.g., housekeeping proteins) for western blotting

These controls help validate antibody specificity and ensure accurate interpretation of experimental results.

How can researchers differentiate between different phosphorylated forms of β-catenin?

Differentiating between various phosphorylated forms of β-catenin requires careful experimental design:

• Use of site-specific phospho-antibodies: In addition to phospho-Ser37 antibodies, researchers can use antibodies specific to other phosphorylation sites (Ser33, Ser45, Thr41) to determine the phosphorylation pattern.

• Sequential immunoprecipitation: Perform IP with one phospho-specific antibody followed by western blotting with another to identify proteins containing multiple phosphorylation sites.

• Mass spectrometry analysis: For comprehensive phosphorylation profiling, mass spectrometry can identify all phosphorylation sites present on β-catenin.

• Genetic approaches: Using cells expressing tagged wild-type and mutant β-catenin (e.g., HCT116 cells with endogenously tagged wild-type and ΔSer45 alleles) can help distinguish between differently phosphorylated forms .

• Temporal analysis: Since phosphorylation at Ser45 precedes phosphorylation at Thr41, Ser37, and Ser33, time-course experiments can reveal the sequence of phosphorylation events .

How can endogenous tagging strategies be used to study β-catenin phosphorylation dynamics?

Endogenous tagging provides powerful tools for studying β-catenin phosphorylation dynamics in their native cellular context. Based on recent research approaches:

• CRISPR/Cas9-mediated gene editing: Researchers can use CRISPR/Cas9 to introduce fluorescent tags at the C-terminal region of β-catenin, as demonstrated in HCT116 colon cancer cells . This approach involves:

  • Identifying suitable sgRNA sequences targeting the C-terminal region

  • Designing donor templates with homology arms close to the sgRNA PAM sequence

  • Incorporating fluorescent proteins (e.g., mClover3 and mCherry2) along with epitope tags (FLAG, V5)

  • Using flexible GS linkers to avoid steric hindrance

• Allele-specific tagging: In heterozygous cell lines (e.g., HCT116 with one wild-type and one ΔSer45 mutant allele), different fluorescent tags can be incorporated into each allele to simultaneously visualize and quantify wild-type and mutant β-catenin .

• Live-cell imaging: The fluorescent tags enable real-time visualization of β-catenin localization, trafficking, and degradation in response to Wnt pathway modulation.

• Quantitative analysis: Techniques such as Fluorescence Correlation Spectroscopy (FCS) can be applied to quantitatively measure the dynamics of tagged β-catenin molecules in living cells following treatments with Wnt pathway modulators .

What methods can be used to study the relationship between Ser37 phosphorylation and β-catenin degradation?

To investigate the relationship between Ser37 phosphorylation and β-catenin degradation, researchers can employ several complementary approaches:

• Pulse-chase experiments: Label β-catenin and track its degradation rate under conditions that promote or inhibit Ser37 phosphorylation.

• Proteasome inhibitors: Use inhibitors like MG132 to block degradation and examine accumulation of phosphorylated β-catenin at Ser37.

• Ubiquitination assays: Evaluate the ubiquitination status of wild-type vs. S37A mutant β-catenin to demonstrate the role of this phosphorylation in targeting for degradation.

• Western blot analysis: Track both full-length and degraded forms of phospho-β-catenin (Ser37) over time following pathway stimulation or inhibition .

• Fluorescence-based degradation reporters: In cells with fluorescently tagged β-catenin, measure fluorescence intensity changes following treatments that affect Ser37 phosphorylation .

• Co-immunoprecipitation: Examine interactions between phosphorylated β-catenin and components of the destruction complex (AXIN, APC, GSK3β) or ubiquitination machinery (BTRC) .

How can Phospho-CTNNB1 (Ser37) antibodies be used to investigate cross-talk between Wnt signaling and other pathways?

Phospho-CTNNB1 (Ser37) antibodies are valuable tools for investigating cross-talk between Wnt signaling and other cellular pathways:

• Dual pathway stimulation/inhibition: Treat cells with modulators of both Wnt and other signaling pathways (e.g., MAPK, PI3K/AKT, Notch), then measure Ser37 phosphorylation status to identify potential cross-regulation.

• Phosphorylation profiling: Compare phosphorylation patterns at multiple sites (including Ser37) following various treatments to identify pathway-specific signatures.

• Combinatorial immunostaining: Perform co-staining with Phospho-CTNNB1 (Ser37) antibodies and markers of other signaling pathways to identify cells with concurrent pathway activation/inhibition.

• Single-cell analysis: In heterogeneous populations or tissues, correlate Ser37 phosphorylation status with activation markers of other pathways at the single-cell level.

• Genetic approaches: Use gene editing to modify components of intersecting pathways and examine effects on β-catenin Ser37 phosphorylation, particularly in the context of post-translational modifications that affect β-catenin function, such as phosphorylation by AMPK at Ser-552, CDK5 at Ser-191/Ser-246, or tyrosine phosphorylation by PTK6 at Tyr-64 .

What are common issues encountered when using Phospho-CTNNB1 (Ser37) antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with Phospho-CTNNB1 (Ser37) antibodies:

• Low signal intensity:

  • Ensure phosphatase inhibitors are included in all buffers

  • Reduce time between sample collection and analysis

  • Optimize antibody concentration and incubation conditions

  • Consider enriching phosphorylated proteins using phospho-protein purification kits

• Non-specific bands in western blots:

  • Increase blocking time and washing steps

  • Titrate antibody concentration

  • Use peptide competition assays to confirm specificity

  • Be aware that multiple band patterns may represent degraded/fragmented forms of phospho-β-catenin rather than non-specific binding

• Inconsistent results between experiments:

  • Standardize cell culture conditions, as confluence levels can affect Wnt signaling

  • Maintain consistent timing for treatments and sample collection

  • Use freshly prepared reagents, especially pathway modulators

  • Consider that endogenous Wnt production may vary between cell batches

• Difficulty detecting phosphorylated β-catenin in vivo or in tissues:

  • Optimize tissue fixation protocols to preserve phospho-epitopes

  • Consider antigen retrieval methods compatible with phospho-epitopes

  • Use signal amplification methods for IHC/IF applications

How should researchers interpret changes in β-catenin Ser37 phosphorylation in the context of Wnt pathway activity?

Interpreting changes in β-catenin Ser37 phosphorylation requires careful consideration of the Wnt signaling context:

• Inverse relationship with pathway activation: In general, increased Ser37 phosphorylation indicates reduced Wnt pathway activity, as this modification targets β-catenin for degradation . Conversely, decreased Ser37 phosphorylation suggests Wnt pathway activation.

• Temporal dynamics: The rapid degradation of phosphorylated β-catenin means that timing is critical for interpretation . Early after Wnt inhibition, there may be a transient increase in detectable phospho-Ser37 before degradation processes reduce levels.

• Subcellular localization: Combine phospho-specific detection with localization studies, as nuclear accumulation of total β-catenin with decreased phospho-Ser37 strongly indicates pathway activation.

• Correlation with downstream targets: Always correlate phosphorylation changes with established readouts of Wnt pathway activity, such as expression of target genes (e.g., AXIN2, c-MYC) or reporter assays (e.g., TOPFlash).

• Multiple phosphorylation sites: Consider that phosphorylation at Ser45 precedes Ser37 phosphorylation , so examining both sites provides more complete information about the β-catenin phosphorylation cascade.

• Allele-specific effects: In cells with β-catenin mutations (e.g., ΔSer45 in HCT116 cells), interpret phosphorylation patterns in the context of which allele is being assessed, as demonstrated in studies using differentially tagged alleles .

What considerations are important when comparing phospho-β-catenin levels across different experimental models?

When comparing phospho-β-catenin levels across different experimental models, several factors should be considered:

• Baseline Wnt activity: Different cell lines and tissues have varying levels of endogenous Wnt pathway activation, affecting baseline phospho-β-catenin levels.

• Genetic background: Check for mutations in β-catenin (CTNNB1) or other Wnt pathway components that might affect phosphorylation patterns. For example, HCT116 cells harbor one wild-type and one ΔSer45 mutant allele of β-catenin .

• Antibody cross-reactivity: Confirm that the antibody shows similar specificity and sensitivity across the species being compared .

• Normalization approach: Determine whether to normalize to total β-catenin levels, housekeeping proteins, or use absolute quantification methods depending on the experimental question.

• Technical variability: Standardize sample preparation, antibody lots, and detection methods across comparisons to minimize technical artifacts.

• Validation with multiple methods: Corroborate phospho-specific antibody results with other techniques such as mass spectrometry or Phos-tag gels when comparing across models.

• Kinetics of responses: Consider that different models may show different temporal dynamics of phosphorylation and degradation following pathway modulation.

How might single-cell analysis techniques advance our understanding of β-catenin phosphorylation heterogeneity?

Single-cell analysis techniques offer promising avenues for understanding the heterogeneity of β-catenin phosphorylation in complex systems:

• Single-cell phosphoproteomics: Emerging technologies that enable phosphoprotein analysis at the single-cell level could reveal cell-to-cell variability in β-catenin phosphorylation states that are masked in bulk analysis.

• Live-cell imaging of endogenously tagged β-catenin: Building on approaches like the CRISPR/Cas9-mediated fluorescent tagging of β-catenin alleles , researchers can track phosphorylation-dependent degradation in individual cells in real-time.

• Mass cytometry (CyTOF): Antibodies against different phosphorylated forms of β-catenin could be used in CyTOF to simultaneously assess multiple post-translational modifications across thousands of individual cells.

• Spatial transcriptomics combined with phospho-protein imaging: Correlating spatial patterns of gene expression with β-catenin phosphorylation status could reveal microenvironmental influences on Wnt signaling activity.

• Microfluidic approaches: Single-cell capture and analysis platforms could allow for dynamic stimulation of individual cells while monitoring phospho-β-catenin responses, revealing cell-specific thresholds and kinetics.

What are emerging techniques for studying the dynamics of β-catenin phosphorylation in live cells?

Emerging techniques for studying β-catenin phosphorylation dynamics in live cells include:

• Phospho-specific FRET sensors: Genetically encoded biosensors that undergo conformational changes upon β-catenin phosphorylation could enable real-time visualization of phosphorylation events.

• Fluorescence Correlation Spectroscopy (FCS): As demonstrated in recent research, FCS allows quantitative measurement of fluorescently tagged β-catenin mobility and interactions in living cells following treatments with Wnt pathway modulators .

• Lattice light-sheet microscopy: This technique enables high-resolution, low-phototoxicity imaging of protein dynamics in 3D over extended periods, ideal for tracking β-catenin movement following phosphorylation.

• Optogenetic control of kinase activity: Combining light-controlled activation of GSK3β with imaging of fluorescently tagged β-catenin could provide precise temporal control over phosphorylation events.

• Fluorescent lifetime imaging microscopy (FLIM): FLIM-based approaches could detect changes in the microenvironment of fluorescently tagged β-catenin associated with phosphorylation status.

• CRISPR-based lineage tracing with β-catenin reporters: This approach could link β-catenin phosphorylation states to cell fate decisions in developmental contexts.

What are the key considerations for designing experiments to investigate β-catenin phosphorylation in different research contexts?

When designing experiments to investigate β-catenin phosphorylation, researchers should consider:

• Research question specificity: Clearly define whether the focus is on detection, quantification, localization, or functional consequences of β-catenin phosphorylation at Ser37.

• Experimental system selection: Choose appropriate models based on endogenous Wnt pathway activity, presence of mutations in pathway components, and technical compatibility with desired assays.

• Technical approach optimization: Select antibodies with validated specificity for phospho-Ser37 , optimize sample preparation to preserve phosphorylation status, and include appropriate controls.

• Temporal considerations: Design time-course experiments that account for the rapid degradation of phosphorylated β-catenin and the sequential nature of phosphorylation events .

• Comprehensive analysis: Combine detection of Ser37 phosphorylation with assessment of other phosphorylation sites and downstream functional readouts of Wnt pathway activity.

• Advanced methodologies: Consider innovative approaches such as endogenous tagging of β-catenin alleles for more physiologically relevant studies of phosphorylation dynamics.

• Cross-disciplinary integration: Incorporate findings into broader cellular contexts by exploring connections between β-catenin phosphorylation and other signaling pathways, cellular processes, or disease mechanisms.