PYGO2 Antibody, HRP conjugated

What is PYGO2 and why is it a significant research target?

PYGO2 (pygopus family PHD finger 2) is a 41.2 kilodalton protein involved in signal transduction through the Wnt pathway . Recent studies have revealed PYGO2's critical role in cancer biology, with genetic deletion studies demonstrating decelerated tumor progression, diminished metastases, and extended survival in experimental models . The protein has been identified as a key orchestrator of immunosuppressive tumor microenvironments through a p53/Sp1/Kit/Ido1 signaling network, making it a promising target for cancer immunotherapy research .

What are the primary applications for HRP-conjugated PYGO2 antibodies?

HRP-conjugated PYGO2 antibodies are optimized for direct detection applications including Western blot (WB), immunohistochemistry on paraffin-embedded tissues (IHC-P), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence (IF) . The horseradish peroxidase conjugation eliminates the need for secondary antibody incubation, allowing for direct visualization through chromogenic or chemiluminescent substrates, which is particularly valuable for reducing background and enhancing detection sensitivity in complex tissue samples .

What are the optimal sample preparation methods for PYGO2 detection?

For optimal PYGO2 detection, tissue lysates should be freshly prepared and used immediately to minimize protein degradation . Recommended protein loading is approximately 20 μg per lane for tissue samples like liver and testis . For blocking, 5% non-fat dry milk in TBST has shown excellent results in reference experiments . When preparing cell or tissue samples, inclusion of protease inhibitors is essential, as is maintaining cold conditions throughout sample processing to preserve protein integrity and epitope accessibility.

What controls should be included when working with PYGO2 antibodies?

Essential controls include positive tissue controls (testis tissue shows strong PYGO2 expression ), negative controls (tissues with minimal PYGO2 expression), loading controls (such as GAPDH at 1/200,000 dilution as referenced in published protocols ), and specificity controls using PYGO2 knockout or knockdown samples where available . For studies involving cancer models, the TS3132, PS and PSP cell lines mentioned in literature provide valuable positive and negative control systems .

How can PYGO2 antibodies be utilized to investigate Wnt signaling pathways?

To investigate Wnt signaling pathways, PYGO2 antibodies can be employed in chromatin immunoprecipitation (ChIP) assays to identify genomic regions bound by PYGO2 in Wnt target gene regulation . Combined immunoprecipitation (IP) followed by Western blot analysis can reveal PYGO2 interactions with other Wnt pathway components . Time-course experiments following Wnt pathway stimulation can track dynamic changes in PYGO2 localization or expression levels, providing insights into pathway activation kinetics and regulatory mechanisms.

What methodologies are effective for studying PYGO2's role in tumor immunology?

For tumor immunology studies, researchers should consider multiplex immunofluorescence approaches combining PYGO2 detection with immune cell markers (e.g., CD8+ T cells) . Flow cytometry with intracellular staining using HRP-conjugated PYGO2 antibodies can quantify expression levels across different cell populations within tumor microenvironments . Genetic ablation models (such as the PSP cell lines) compared with PYGO2-expressing counterparts (PS cells) allow assessment of how PYGO2 affects immune cell infiltration and activation . Immunodepletion experiments using anti-CD8 neutralizing antibodies can confirm the mechanism of PYGO2's impact on tumor growth through T-cell regulation .

What dilution ranges and incubation conditions yield optimal results with HRP-conjugated PYGO2 antibodies?

For Western blot applications, a 1:1000 dilution has been validated for specific detection of PYGO2 protein . The antibody works efficiently in standard high phosphate PBS buffer (100 mM phosphate, 150 mM NaCl, pH 7.6) . Optimal incubation conditions typically involve overnight incubation at 4°C for primary antibody steps, though specific protocols may vary based on sample type and detection system. When using HRP-conjugated antibodies, shorter incubation times may be possible due to the direct detection capability.

How can background signal be minimized when using HRP-conjugated PYGO2 antibodies?

To reduce background signal, researchers should: (1) block endogenous peroxidase activity with hydrogen peroxide treatment prior to antibody application, (2) optimize blocking conditions using different agents at various concentrations, (3) include appropriate detergents in wash buffers, (4) titrate antibody concentration to determine the optimal signal-to-noise ratio, and (5) increase washing duration and frequency between steps. For tissues with high background, a specialized blocking protocol may be necessary, potentially including avidin/biotin blocking for tissues with high endogenous biotin.

How does PYGO2 expression correlate with tumor progression and immunotherapy response?

Research indicates that PYGO2 expression is inversely correlated with CD8+ T cell infiltration in human prostate cancer samples . In experimental models, genetic ablation or pharmacological inhibition of PYGO2 has been shown to sensitize prostate cancer cells to immune checkpoint blockade, adoptive T-cell therapy, and myeloid-derived suppressor cell inhibition . The research methodology involves comparative analysis of tumor growth kinetics between PYGO2-expressing and PYGO2-knockout models in both immune-competent and immune-deficient backgrounds, revealing the protein's cell non-autonomous activity in restricting effector T cell infiltration and cytotoxicity .

What methodological approaches are recommended for analyzing PYGO2's impact on metastatic potential?

To assess PYGO2's role in metastasis, researchers have employed intracardiac injection models with luciferase-labeled cells (such as the tk-GFP-luciferase reporter system) to track metastatic spread to bone, lungs, liver, and brain . Colony formation assays provide quantitative data on cell-autonomous effects of PYGO2 on cancer cell proliferation and survival . For in vivo assessment, subcutaneous tumor growth models in both immune-competent and immune-deficient backgrounds help distinguish between PYGO2's intrinsic effects on tumor cells versus its immunomodulatory functions .

How should researchers interpret variations in PYGO2 detection across different tissue types?

When analyzing PYGO2 expression across tissues, consider that expression levels vary significantly between tissue types, with testis showing notably higher expression than liver in both mouse and rat samples . These differences may reflect tissue-specific roles of PYGO2 in development and homeostasis. Quantitative analysis should normalize PYGO2 signal to appropriate loading controls and include statistical comparisons across multiple biological replicates. Researchers should also consider potential post-translational modifications that might affect antibody recognition in different tissue contexts.

What are approaches for validating antibody specificity in PYGO2 research?

For rigorous validation of PYGO2 antibody specificity, researchers should: (1) compare staining patterns across multiple antibodies targeting different PYGO2 epitopes, (2) include genetic PYGO2 knockout controls such as CRISPR/Cas9-modified cell lines , (3) perform peptide competition assays using the immunizing peptide, (4) validate with orthogonal techniques such as RNA expression analysis, and (5) confirm the molecular weight of detected bands (approximately 41.2 kDa for PYGO2) . The established knockout models described in the literature, including the PSP and PYGO2-knockout RM9 cell lines, provide valuable tools for antibody validation .

How can PYGO2 antibodies be employed in studying combination immunotherapy approaches?

Building on findings that PYGO2 deletion enhances T cell infiltration and sensitizes tumors to T cell killing , researchers can use HRP-conjugated PYGO2 antibodies to monitor expression changes during immunotherapy treatment. Methodological approaches include multiplex immunohistochemistry to simultaneously visualize PYGO2 and immune markers, analysis of pre- and post-treatment biopsies to track PYGO2 levels, and correlation studies between PYGO2 expression and response to various immunotherapeutic agents. These approaches support the development of combination strategies targeting PYGO2 alongside existing immunotherapies.

What methodologies are recommended for investigating PYGO2's mechanistic role in the p53/Sp1/Kit/Ido1 signaling network?

To dissect PYGO2's role in the p53/Sp1/Kit/Ido1 signaling network, researchers should employ chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify direct genomic targets of PYGO2 . Co-immunoprecipitation studies can confirm protein-protein interactions within this pathway. Gene expression analysis comparing wild-type and PYGO2-knockout models can reveal downstream effects on signaling components. Pharmacological inhibition studies targeting different nodes in this network can help establish the hierarchy and interdependence of these factors in creating immunosuppressive tumor microenvironments .

How can researchers address inconsistent PYGO2 detection in Western blot applications?

When encountering inconsistent PYGO2 detection in Western blots, consider these methodological solutions: (1) ensure lysates are freshly prepared and used immediately to prevent protein degradation , (2) optimize protein loading (20 μg per lane has been validated in reference protocols) , (3) verify transfer efficiency with reversible protein staining, (4) adjust antibody dilution (1:1000 is recommended based on validated protocols) , and (5) extend exposure time for detecting low-abundance signals. If bands appear at unexpected molecular weights, consider the presence of splice variants, post-translational modifications, or potential degradation products.