Phospho-CTNNB1 (Tyr489) Antibody

What is Phospho-CTNNB1 (Tyr489) Antibody and what specifically does it detect?

The Phospho-CTNNB1 (Tyr489) Antibody is a polyclonal antibody that specifically detects endogenous levels of Catenin Beta-1 (β-catenin) protein only when phosphorylated at the Tyrosine 489 residue. It does not recognize the unphosphorylated form or β-catenin phosphorylated at other sites. This high specificity makes it valuable for studying this particular post-translational modification in β-catenin signaling pathways .

The antibody is typically generated in rabbits using a synthetic phosphopeptide corresponding to amino acid residues surrounding the Tyr489 phosphorylation site. The immunogen peptide sequence commonly used is derived from human β-catenin around the phosphorylation site (with the core sequence L-H-Y(p)-G-L), which is conserved across multiple species including human, mouse, and rat .

What applications is the Phospho-CTNNB1 (Tyr489) Antibody validated for?

The Phospho-CTNNB1 (Tyr489) Antibody has been validated for multiple research applications, with specific protocols optimized for each technique:

The antibody has confirmed reactivity with human, mouse, rat, and monkey samples, making it versatile for comparative studies across species .

What are the optimal storage and handling conditions for maintaining antibody performance?

For maximum stability and performance, the Phospho-CTNNB1 (Tyr489) Antibody should be:

• Stored at -20°C for up to one year from the date of receipt

• Aliquoted upon first thawing to minimize repeated freeze-thaw cycles

• Maintained in its storage buffer, typically PBS (pH 7.4) containing 50% glycerol, small amounts of protein stabilizers (0.5% BSA), and 0.02% sodium azide as a preservative

• Thawed completely before use and mixed gently to ensure homogeneity

• Never stored at room temperature for extended periods

Repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and loss of antibody activity .

How should I design controls for experiments using Phospho-CTNNB1 (Tyr489) Antibody?

Robust experimental design with appropriate controls is critical when working with phospho-specific antibodies:

Positive controls:

• Cell lysates from cells treated with growth factors known to induce Tyr489 phosphorylation (e.g., EGF, which stimulates tyrosine phosphorylation of β-catenin)

• Recombinant phosphorylated proteins when available

Negative controls:

• Lysates treated with phosphatase to remove phosphorylation

• Lysates from cells where CTNNB1 expression is knocked down

• Blocking with the immunizing phosphopeptide to demonstrate specificity

• Use of non-phospho-specific β-catenin antibody in parallel to compare total protein levels

Technical controls:

• Loading controls (GAPDH or similar housekeeping proteins) for normalization in Western blots

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

What are the recommended protocols for detecting phospho-β-catenin (Tyr489) in tissue sections?

For immunohistochemical detection in tissue sections:

• Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Block endogenous peroxidase activity using 3% hydrogen peroxide

• Apply protein blocking solution to reduce non-specific binding

• Incubate with primary Phospho-CTNNB1 (Tyr489) Antibody at 1:100-1:300 dilution overnight at 4°C

• Wash thoroughly with TBS buffer

• Apply biotinylated secondary antibody followed by streptavidin-HRP

• Develop with chromogen (e.g., DAB)

• Counterstain with hematoxylin

• Evaluate staining in different subcellular compartments (membrane, cytoplasm, nucleus) using a scoring system:

  • 0: No staining

  • 1: Weak staining

  • 2: Moderate staining

  • 3: Strong staining

How can I optimize Western blot protocols for phospho-β-catenin (Tyr489) detection?

For optimal Western blot results:

• Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride, etc.) in all buffers during sample preparation to preserve phosphorylation status

• Use fresh samples whenever possible; phosphorylation can be labile

• Load adequate protein (25-50 μg total protein per lane)

• Transfer proteins to PVDF membrane (preferred over nitrocellulose for phosphoproteins)

• Block with 5% BSA in TBST (not milk, which contains phosphoproteins that can interfere)

• Incubate with Phospho-CTNNB1 (Tyr489) Antibody at 1:500-1:1000 dilution overnight at 4°C

• Wash thoroughly with TBST (at least 3×10 minutes)

• Use high-sensitivity ECL detection systems for visualization

• Strip and reprobe with total β-catenin antibody to normalize phospho-signal to total protein levels

What is the significance of Tyr489 phosphorylation in β-catenin signaling?

Tyrosine 489 phosphorylation represents an important regulatory mechanism in β-catenin function that differs from the well-characterized N-terminal serine/threonine phosphorylation:

• While N-terminal phosphorylation (Ser33/Ser37/Thr41) by GSK3β typically leads to β-catenin degradation, tyrosine phosphorylation often influences protein-protein interactions and subcellular localization

• Tyr489 phosphorylation may affect β-catenin's interaction with cadherins and its role in cell adhesion

• EGF and other growth factors can stimulate tyrosine phosphorylation of β-catenin, potentially providing crosstalk between growth factor signaling and Wnt pathway

• Unlike N-terminal phosphorylated β-catenin which is primarily marked for degradation, Tyr489 phosphorylated β-catenin may have distinct functions in transcriptional regulation and cell adhesion

This makes Tyr489 phosphorylation detection particularly valuable for understanding the dual role of β-catenin in adhesion and transcription.

How does the subcellular distribution of phospho-β-catenin (Tyr489) compare between normal and cancer tissues?

Research using phospho-specific antibodies has revealed distinct patterns of phospho-β-catenin distribution:

• In colorectal cancer patient samples, phospho-β-catenin (Ser33/Ser37/Thr41) shows primarily nuclear (25.8% of cases) or cytoplasmic (18.8% of cases) localization, with only a small subset showing both patterns

• Approximately 10.8% of colorectal cancer cases show moderate to strong nuclear staining for phospho-β-catenin

• More than 50% of colorectal cancer cases show neither nuclear nor cytoplasmic staining for N-terminally phosphorylated β-catenin

• In normal colon epithelium, phospho-β-catenin staining is generally weak or absent

• The distinct localization patterns may reflect different pathological mechanisms and could potentially serve as prognostic indicators

While these observations are based on studies of N-terminal phosphorylated β-catenin, similar analyses with Tyr489 phospho-specific antibodies would provide valuable comparative data on different phosphorylation states in cancer progression.

How can Phospho-CTNNB1 (Tyr489) Antibody be used in cell-based phosphorylation assays?

Cell-based phosphorylation ELISA assays provide an efficient method for monitoring changes in CTNNB1 phosphorylation in response to various stimuli:

• Plate cells in 96-well format and treat with test compounds or stimuli

• Fix cells with 4% paraformaldehyde to preserve phosphorylation state

• Permeabilize with 0.1% Triton X-100

• Block with appropriate blocking buffer

• Incubate with Phospho-CTNNB1 (Tyr489) Antibody

• Apply HRP-conjugated secondary antibody

• Develop with colorimetric substrate

• Measure absorbance using a plate reader

• Normalize data to total protein content or cell number using parallel wells stained for total β-catenin or GAPDH

This approach allows for high-throughput screening of compounds that might influence β-catenin phosphorylation status .

What should I consider when interpreting immunostaining patterns with Phospho-CTNNB1 (Tyr489) Antibody?

When analyzing immunostaining results, consider these important factors:

• Subcellular localization - Different phosphorylation states of β-catenin may show distinct localization patterns:

  • Membrane localization typically indicates association with cell adhesion complexes

  • Cytoplasmic accumulation may represent protein not engaged in either adhesion or transcription

  • Nuclear localization suggests potential involvement in transcriptional regulation

• Staining intensity gradients - In normal tissues, look for:

  • Gradient patterns in epithelial tissues that may correlate with differentiation status

  • Changes in staining patterns at tissue boundaries or in specific cell types

• Heterogeneity in tumor samples - Be aware that:

  • Different regions of a tumor may show variable staining

  • Score based on the predominant pattern, but note heterogeneity

  • Compare with adjacent normal tissue when possible

• Correlation with other markers - Consider parallel staining for:

  • Total β-catenin to understand the ratio of phosphorylated to total protein

  • Other Wnt pathway components to establish context

  • Proliferation markers like Ki-67 to correlate with functional outcomes

What are common issues when using Phospho-CTNNB1 (Tyr489) Antibody and how can they be resolved?

When troubleshooting, always run appropriate positive and negative controls to establish a baseline for comparison .

How can I validate the specificity of Phospho-CTNNB1 (Tyr489) Antibody in my experimental system?

To confirm antibody specificity:

• Peptide competition assay:

  • Pre-incubate the antibody with excess phospho-peptide immunogen

  • A genuine signal should be blocked by the phospho-peptide

  • Include a non-phosphorylated peptide control that should not block the signal

• Phosphatase treatment:

  • Treat one sample set with lambda phosphatase

  • Signal should disappear in phosphatase-treated samples

• Genetic validation:

  • Use cells with CTNNB1 knockout or knockdown

  • Alternatively, use cells expressing a Tyr489 mutant (Y489F)

  • Signal should be absent in these samples

• Treatment with kinase activators/inhibitors:

  • Treat cells with compounds known to modulate the kinases responsible for Tyr489 phosphorylation

  • Monitor corresponding changes in signal intensity

What considerations are important when selecting between different anti-CTNNB1 antibodies for a research project?

When choosing between different CTNNB1 antibodies, consider:

• Phosphorylation site specificity:

  • Different phosphorylation sites (Tyr489, Tyr654, Ser33/37/Thr41, etc.) have distinct functional implications

  • Choose based on the specific pathway or mechanism being studied

• Antibody format:

  • Monoclonal antibodies offer high reproducibility and specificity for a single epitope

  • Polyclonal antibodies may provide stronger signals but with potential for more background

• Host species:

  • Consider compatibility with other antibodies in multi-labeling experiments

  • Rabbit-derived antibodies (like most Phospho-CTNNB1 (Tyr489) antibodies) generally work well for most applications

• Validated applications:

  • Ensure the antibody is validated for your specific application (WB, IHC, IF, ELISA)

  • Review literature using the same antibody for similar applications

• Species reactivity:

  • Confirm cross-reactivity with your experimental model species

  • The Phospho-CTNNB1 (Tyr489) antibody typically reacts with human, mouse, rat, and sometimes monkey samples

How does Tyr489 phosphorylation relate to other β-catenin phosphorylation events?

β-catenin is regulated by a complex network of phosphorylation events that determine its stability, localization, and function:

• N-terminal phosphorylation (Ser33, Ser37, Thr41) by GSK3β following priming phosphorylation at Ser45 targets β-catenin for ubiquitination and proteasomal degradation

• Ser552 phosphorylation by AMPK promotes protein stabilization and enhances TCF/LEF-mediated transcription

• Ser191 and Ser246 phosphorylation by CDK5 affects β-catenin function

• Tyrosine phosphorylation sites including Tyr489 are often targets of growth factor signaling:

  • EGF stimulates tyrosine phosphorylation

  • PTK6 phosphorylates β-catenin at multiple tyrosine residues (Tyr64, Tyr142, Tyr331, Tyr333)

  • These tyrosine phosphorylation events may regulate β-catenin's role in cell adhesion versus transcriptional activity

Understanding the interplay between these different phosphorylation events is crucial for deciphering β-catenin's role in normal physiology and disease.

What is the relevance of studying phospho-β-catenin (Tyr489) in cancer research?

Studying phospho-β-catenin (Tyr489) in cancer offers several important insights:

• Diagnostic and prognostic value:

  • Altered patterns of β-catenin phosphorylation may serve as biomarkers

  • Studies show distinct subcellular distribution patterns of phospho-β-catenin in cancer versus normal tissues

• Therapeutic targeting:

  • Understanding specific phosphorylation events may reveal novel therapeutic targets

  • CTNNB1 mutations drive cancer progression in colorectal, liver, and ovarian cancers

• Pathway crosstalk:

  • Tyrosine phosphorylation often links growth factor signaling to Wnt pathway activity

  • This crosstalk may explain resistance mechanisms to targeted therapies

• Functional differences:

  • Different phosphorylation states may explain the dual role of β-catenin in adhesion versus transcription

  • Changes in phosphorylation patterns may precede genetic alterations during cancer progression

The ability to specifically detect Tyr489 phosphorylation provides researchers with a powerful tool to investigate these aspects of cancer biology.