Phospho-YWHAZ (Ser58) Antibody

What is YWHAZ and why is phosphorylation at Ser58 significant?

YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta) is a member of the highly conserved 14-3-3 protein family that mediates signal transduction by binding to phosphoserine-containing proteins. This adapter protein regulates a broad spectrum of both general and specialized signaling pathways through interactions with numerous binding partners, typically through recognition of phosphoserine or phosphothreonine motifs . The protein is 99% identical across mouse, rat, and sheep orthologs, highlighting its evolutionary importance .

Ser58 phosphorylation is particularly significant as it serves as a regulatory switch that can modulate YWHAZ function in various cellular processes. This specific phosphorylation site affects the protein's ability to bind partners and influences its role in signaling cascades related to cellular proliferation, migration, and differentiation .

What experimental applications are suitable for Phospho-YWHAZ (Ser58) antibody?

Phospho-YWHAZ (Ser58) antibody is validated for multiple research applications:

These applications enable researchers to investigate YWHAZ phosphorylation status in multiple experimental contexts . The antibody specifically detects endogenous levels of 14-3-3 Zeta protein only when phosphorylated at Ser58, making it valuable for studying this specific post-translational modification .

How should Phospho-YWHAZ (Ser58) antibody be stored and handled?

For optimal performance and longevity, store the antibody at -20°C for up to 1 year from the date of receipt. Repeated freeze-thaw cycles should be avoided as they can compromise antibody integrity. The antibody is typically formulated as a liquid in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide . When handling the antibody:

• Aliquot upon first thaw to minimize freeze-thaw cycles

• Thaw completely before use and mix gently

• Brief centrifugation may be needed if precipitation occurs

• Return unused portions to -20°C immediately after use

• Note that the antibody is for research use only (RUO) and must not be used in diagnostic or therapeutic applications

How can I validate the specificity of Phospho-YWHAZ (Ser58) antibody for my research?

Validating antibody specificity is crucial for phospho-specific antibodies. A comprehensive validation approach includes:

• Western blot with control treatments: Compare samples from cells treated with phosphatase inhibitors versus phosphatase enzymes. The signal should disappear after phosphatase treatment if the antibody is truly phospho-specific .

• Stimulation-inhibition experiments: Apply stimuli known to induce Ser58 phosphorylation and inhibitors of relevant kinases. This creates positive and negative controls for phosphorylation status .

• Peptide competition assays: Pre-incubate the antibody with phosphorylated and non-phosphorylated peptides containing the Ser58 site. Only the phosphorylated peptide should block antibody binding .

• Genetic controls: Use YWHAZ knockout models or cells with Ser58 mutations (S58A) as negative controls. These approaches are particularly robust for confirming specificity .

• Cross-reactivity assessment: Test reactivity against other 14-3-3 family members to ensure the antibody specifically recognizes phosphorylated YWHAZ and not related proteins .

The most rigorous validation combines multiple approaches to firmly establish antibody specificity in your experimental system .

What are the optimal antigen retrieval methods for Phospho-YWHAZ (Ser58) immunohistochemistry?

Phosphoepitopes are notoriously sensitive to fixation and processing conditions. For optimal immunohistochemical detection of Phospho-YWHAZ (Ser58):

• Heat-induced epitope retrieval (HIER): Use citrate buffer (pH 6.0) or EDTA buffer (pH 8.0-9.0) with careful optimization for your specific tissue type. HIER is generally more effective than enzymatic methods for phosphoepitopes .

• Pressure cooking vs. microwave: Pressure cooking often provides more consistent results for phosphoepitopes compared to microwave methods.

• Fixation considerations: Overfixation can mask phosphoepitopes. Limit fixation time with formalin to 24 hours when possible.

• Phosphatase inhibitors: Include phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) in buffers during tissue processing and staining to preserve phosphorylation status.

• Signal amplification: Consider tyramide signal amplification or other methods to enhance detection of low-abundance phosphoproteins .

Since standardized protocols for Phospho-YWHAZ (Ser58) antibody may vary between manufacturers, optimize these conditions for your specific experimental system and antibody source.

What role does YWHAZ phosphorylation play in neurodevelopmental disorders?

YWHAZ has emerging significance in neurodevelopmental conditions. Recent research demonstrates that:

• YWHAZ variants are associated with intellectual disability (ID) and global developmental delay (GDD). Whole-exome sequencing identified pathogenic YWHAZ variants in families with these conditions .

• YWHAZ is intolerant to both loss-of-function (pLI = 0.94) and missense (Z = 3.1) variants based on gnomAD data, suggesting its critical importance in neurodevelopment .

• Neurodevelopmental defects have been observed in YWHAZ-deficient mice, supporting its causative role in human disorders .

• Phosphorylation at Ser58 may regulate YWHAZ's interaction with neuronal proteins involved in synapse formation and maturation. The protein regulates spine maturation through modulation of ARHGEF7 activity .

• YWHAZ phosphorylation status potentially influences brain-specific signaling pathways critical for neuronal migration, differentiation, and function.

Researchers investigating neurodevelopmental disorders should consider examining YWHAZ phosphorylation status as a potential biomarker or mechanistic contributor to pathogenesis .

How can Phospho-YWHAZ (Ser58) antibody be used to study cancer progression mechanisms?

Phospho-YWHAZ (Ser58) antibody offers valuable insights into cancer research:

• Metastasis studies: YWHAZ has been identified as a key regulator of pancreatic cancer metastasis. Overexpression results in more aggressive metastatic phenotypes by modulating epithelial-to-mesenchymal transition (EMT) . Phospho-specific antibodies can track how phosphorylation status correlates with metastatic behavior.

• Therapeutic efficacy assessment: Phospho-YWHAZ antibodies can directly demonstrate the efficacy of kinase-targeted cancer therapies that might affect YWHAZ phosphorylation status .

• Signaling pathway analysis: Use the antibody to investigate how cancer-related signaling affects YWHAZ phosphorylation. This can be particularly relevant for pathways involving ERK1/2, as YWHAZ promotes EMT through elevated ERK1/2 phosphorylation in hepatocellular carcinoma .

• Tissue microarray screening: Screen cancer tissue microarrays to establish correlations between Ser58 phosphorylation levels and clinical outcomes or cancer subtypes.

• Single-cell analysis: Combine with single-cell techniques to identify heterogeneity in YWHAZ phosphorylation within tumors.

These approaches can help elucidate the mechanistic roles of phosphorylated YWHAZ in cancer progression and potentially identify new therapeutic targets .

How do I troubleshoot false-negative results when using Phospho-YWHAZ (Ser58) antibody?

False-negative results are a common challenge with phospho-specific antibodies. To troubleshoot:

• Preserve phosphorylation status: Include phosphatase inhibitors in all buffers throughout sample preparation. Common inhibitors include sodium fluoride (50 mM), sodium orthovanadate (1 mM), and phosphatase inhibitor cocktails .

• Optimize fixation: Overfixation can mask phosphoepitopes. For tissues, limit formalin fixation time to 24 hours when possible. For cells, shorter fixation times (10-15 minutes) with 4% paraformaldehyde may preserve epitopes better.

• Test multiple antigen retrieval methods: Systematically compare different antigen retrieval techniques (heat-induced with varying buffers and pH levels) to determine optimal conditions for your samples .

• Increase antibody concentration: If signal is weak, try a higher antibody concentration. For IHC, adjust from 1:100 to 1:50 or even 1:25 if background remains acceptable .

• Verify antibody functionality: Run a positive control sample known to contain phosphorylated YWHAZ (e.g., cells treated with appropriate stimuli) alongside your experimental samples.

• Consider signal amplification: For low-abundance phosphoproteins, employ signal amplification techniques like tyramide signal amplification or polymer detection systems .

If false-negative results persist despite these measures, consider whether the phosphorylation site might be occluded by protein-protein interactions in your specific experimental context.

What signaling pathways involve YWHAZ phosphorylation at Ser58?

Phosphorylation of YWHAZ at Ser58 intersects with multiple signaling networks:

• Insulin signaling: YWHAZ interacts with IRS1 protein, suggesting a role in regulating insulin sensitivity. Ser58 phosphorylation may modulate this interaction .

• Transcriptional regulation: Phosphorylated YWHAZ promotes cytosolic retention and inactivation of TFEB transcription factor by binding to phosphorylated TFEB .

• Cytoskeletal organization: YWHAZ induces ARHGEF7 activity on RAC1, affecting lamellipodia and membrane ruffle formation. Ser58 phosphorylation likely regulates this function .

• Neuronal signaling: In neurons, YWHAZ regulates spine maturation through modulation of ARHGEF7 activity. Phosphorylation at Ser58 may serve as a switch for this function .

• Cancer-related pathways: YWHAZ can promote epithelial-mesenchymal transition through elevated ERK1/2 phosphorylation in hepatocellular carcinoma and inhibit Cdc2 phosphorylation in lung cancer cells .

Understanding how Ser58 phosphorylation affects these pathways requires investigation with phospho-specific antibodies to correlate YWHAZ phosphorylation status with pathway activation.

How does Phospho-YWHAZ (Ser58) antibody compare to other phosphorylation state-specific antibodies in experimental design?

When designing experiments with Phospho-YWHAZ (Ser58) antibody, consider these comparative aspects:

• Epitope stability: Phosphoserines are generally more stable than phosphotyrosines, but less stable than phosphothreonines. Phospho-YWHAZ (Ser58) epitopes may require more careful handling than some other phospho-epitopes .

• Background signal: Unlike generic anti-phosphotyrosine antibodies that can detect multiple phosphoproteins, phospho-YWHAZ antibodies are sequence-specific, typically resulting in cleaner signals with fewer non-specific bands .

• Validation requirements: As with all PSSAs, rigorous validation is essential. For Phospho-YWHAZ (Ser58), this includes demonstrating phosphorylation-dependent recognition and ensuring specificity among 14-3-3 family members .

• Multiplexing potential: Consider combining Phospho-YWHAZ (Ser58) antibody with antibodies against total YWHAZ or other pathway components in multiplexed assays to obtain more comprehensive data in a single experiment.

• Cross-species applicability: The high conservation of YWHAZ across species (99% identity between human, mouse, rat, and sheep) makes this antibody valuable for comparative studies across model organisms .

These considerations should inform experimental design decisions when working with Phospho-YWHAZ (Ser58) antibody in relation to other phospho-specific antibodies.

What are best practices for quantifying Phospho-YWHAZ (Ser58) levels in research samples?

Accurate quantification of Phospho-YWHAZ (Ser58) requires attention to several methodological details:

• Normalization strategy: Always normalize phospho-YWHAZ signal to total YWHAZ levels rather than housekeeping proteins to account for variations in total protein expression. This is best achieved using parallel samples or strip-and-reprobe approaches.

• Standard curve inclusion: For ELISA-based quantification, include a standard curve using recombinant phosphorylated YWHAZ or synthetic phosphopeptides when absolute quantification is needed .

• Phosphorylation ratio calculation: Express results as the ratio of phosphorylated to total YWHAZ, which provides more meaningful biological insights than absolute phospho-signal alone.

• Technical replicates: Include at least three technical replicates per biological sample to account for assay variability.

• Image analysis for IHC/IF: For tissue or cell staining, use digital image analysis with appropriate software to quantify staining intensity in defined regions of interest. Consider using H-score or Allred scoring systems for semi-quantitative assessment.

• Temporal considerations: Phosphorylation states can change rapidly. Standardize sample collection times and processing procedures to minimize variability from temporal fluctuations.

These approaches will help ensure reliable quantification of Phospho-YWHAZ (Ser58) across experimental samples.

How can I design experiments to study the effects of YWHAZ Ser58 phosphorylation on protein-protein interactions?

To investigate how Ser58 phosphorylation affects YWHAZ protein interactions:

• Co-immunoprecipitation with phospho-specific antibodies: Use Phospho-YWHAZ (Ser58) antibody for immunoprecipitation followed by mass spectrometry or Western blotting to identify interaction partners specific to the phosphorylated state.

• Phosphomimetic and phospho-deficient mutants: Generate S58D (phosphomimetic) and S58A (phospho-deficient) YWHAZ mutants for comparative interaction studies. These tools can help establish causality between phosphorylation and specific protein interactions.

• Proximity ligation assay (PLA): Combine Phospho-YWHAZ (Ser58) antibody with antibodies against suspected interaction partners in PLA to visualize interactions in situ with subcellular resolution.

• FRET/BRET analysis: Use fluorescence or bioluminescence resonance energy transfer techniques with tagged YWHAZ variants to monitor real-time interaction dynamics in live cells.

• Peptide array screening: Screen peptide arrays derived from potential binding partners with recombinant phosphorylated and non-phosphorylated YWHAZ to map interaction interfaces.

These approaches can systematically characterize how Ser58 phosphorylation modulates YWHAZ's extensive interactome, particularly with proteins involved in signaling cascades and neuronal functions .

How can Phospho-YWHAZ (Ser58) antibody contribute to neurodevelopmental disorder research?

Phospho-YWHAZ (Ser58) antibody offers several applications for neurodevelopmental research:

• Patient sample analysis: Compare Ser58 phosphorylation levels in accessible patient samples (like blood cells or induced pluripotent stem cell-derived neurons) from individuals with neurodevelopmental disorders versus controls.

• Animal model characterization: Evaluate Ser58 phosphorylation patterns in brain regions of animal models of intellectual disability, global developmental delay, or schizophrenia to identify pathological alterations .

• Developmental timeline studies: Map changes in YWHAZ phosphorylation during neural development to identify critical periods where dysregulation might contribute to neurodevelopmental disorders.

• Drug screening: Screen compounds for their ability to modulate YWHAZ phosphorylation as potential therapeutic approaches for disorders linked to YWHAZ dysfunction.

• Pathway analysis: Determine how disease-associated YWHAZ variants affect Ser58 phosphorylation and downstream signaling in neuronal cells.

These approaches can help elucidate how alterations in YWHAZ phosphorylation contribute to neurodevelopmental pathologies and potentially identify new therapeutic targets .

What controls should be included when using Phospho-YWHAZ (Ser58) antibody in cancer research?

For rigorous cancer research applications, include these controls:

• Positive tissue controls: Use pancreatic cancer samples, which have demonstrated high YWHAZ expression and phosphorylation levels in research studies .

• Treatment controls: Include samples from cells treated with phosphatase inhibitors (positive control) and samples treated with relevant kinase inhibitors (negative control).

• Genetic controls: Use cell lines with YWHAZ knockdown/knockout or cells expressing S58A mutant as negative controls to validate antibody specificity.

• Comparative normal tissue: Include matched normal tissue adjacent to tumor samples to establish baseline phosphorylation levels.

• Isotype controls: For immunohistochemistry or flow cytometry, include appropriate rabbit IgG isotype controls at the same concentration as the primary antibody.

• Competition controls: Pre-incubate the antibody with phosphorylated and non-phosphorylated peptides to demonstrate phospho-specificity in your cancer model.

These controls will strengthen the validity of findings regarding YWHAZ phosphorylation status in cancer progression and metastasis studies .

How can I integrate Phospho-YWHAZ (Ser58) analysis into multi-omics research approaches?

To incorporate Phospho-YWHAZ (Ser58) analysis into comprehensive multi-omics studies: