PYGO2 Antibody, FITC conjugated

What is PYGO2 and what cellular pathways is it involved in?

PYGO2 is a protein-coding gene associated with G-protein coupled receptor (GPCR) and Wnt signaling pathways. It functions in chromatin binding and acts as a histone acetyltransferase regulator . As an emerging epigenetic switch, PYGO2 regulates stem cell self-renewal, somatic cell division, and hormone-induced gene expression through both Wnt-dependent and Wnt-independent pathways . Its role in signal transduction through the Wnt pathway makes it particularly relevant in developmental and cancer biology research .

PYGO2 has both cell-autonomous functions and cell non-autonomous roles in shaping the tumor microenvironment (TME), particularly affecting T-cell infiltration and activity in certain cancers . This dual functionality makes it an intriguing target for both basic science investigations and translational research efforts in oncology.

What applications are most suitable for PYGO2 antibody, FITC conjugated?

The FITC-conjugated PYGO2 antibody is particularly valuable for fluorescence-based applications including:

• Flow cytometry (intracellular): Allows quantitative analysis of PYGO2 expression at the single-cell level

• Immunocytochemistry/Immunofluorescence (ICC/IF): Enables visualization of PYGO2 subcellular localization

• ELISA: Provides quantitative measurement of PYGO2 in biological samples

For researchers studying PYGO2 expression patterns, the FITC conjugation eliminates the need for secondary antibody incubation steps, reducing background and simplifying experimental workflows. The antibody has been validated with human samples and is suitable for detecting PYGO2 protein in its native conformation .

What is the subcellular localization pattern of PYGO2 and how does this affect antibody selection?

PYGO2 demonstrates a complex subcellular distribution pattern with cytoplasmic, membranous, and nuclear localization . This multi-compartment distribution necessitates careful consideration when selecting fixation and permeabilization protocols for immunostaining experiments.

For optimal detection across all subcellular compartments:

• Use paraformaldehyde fixation (4%) followed by gentle detergent permeabilization

• Include both nuclear and cytoplasmic markers in multiplexed imaging experiments

• Consider subcellular fractionation followed by western blotting for quantitative assessment of PYGO2 distribution

The FITC-conjugated antibody is particularly advantageous for visualizing this distribution pattern as its direct fluorescence enables high-resolution imaging of differential localization patterns across experimental conditions .

What are the optimal storage conditions for maintaining PYGO2 antibody, FITC conjugated activity?

To maintain optimal activity of the FITC-conjugated PYGO2 antibody:

• Store at -20°C or -80°C immediately upon receipt

• Avoid repeated freeze-thaw cycles that can degrade both antibody function and FITC fluorescence

• Store in the dark to prevent photobleaching of the FITC conjugate

• Consider aliquoting the antibody into single-use volumes before freezing

• The antibody is typically supplied in a buffer containing 50% glycerol and PBS (pH 7.4) with 0.03% Proclin 300 as a preservative

When preparing working dilutions, maintain temperature at 4°C and use within 24 hours for optimal results. Long-term storage of diluted antibody is not recommended due to potential loss of activity and fluorescence intensity.

What positive control tissues are recommended for validating PYGO2 antibody performance?

Based on validated expression patterns, the following tissues serve as excellent positive controls for PYGO2 antibody validation:

Transitional cell carcinoma samples have also been validated as reliable positive controls . For negative controls, consider using tissue known to express low PYGO2 levels or employ isotype-matched, non-specific antibodies to establish background staining levels.

How can PYGO2 antibody be used to investigate cancer progression mechanisms?

PYGO2 has significant associations with cancer progression across multiple tumor types. The FITC-conjugated antibody can be employed in several advanced experimental designs:

• Tumor Microenvironment Studies: Research has revealed that PYGO2 plays a cell non-autonomous role in shaping the immunosuppressive tumor microenvironment of prostate cancer, particularly affecting T-cell infiltration and activity . Flow cytometry with the FITC-conjugated antibody allows evaluation of PYGO2 expression in various cell populations within the tumor microenvironment simultaneously.

• Cancer Grade Correlation: Studies have shown that 59% of patient tumor specimens exhibit positive PYGO2 immunohistochemistry staining with increased intensity correlating with the grade of malignancy, especially for WHO grade III and IV tumors . The FITC-conjugated antibody can be used in tissue microarray studies to systematically evaluate this correlation across larger patient cohorts.

• Metastasis Investigation: Significant associations have been found between PYGO2 protein expression in colorectal cancer and tumor cell metastasis to lymph nodes . Using the antibody in longitudinal studies of primary and metastatic samples can help elucidate the mechanistic role of PYGO2 in the metastatic cascade.

• Prognostic Biomarker Development: Abnormal PYGO2 expression has been associated with poor differentiation, advanced tumor stage, and poor prognosis in non-small cell lung cancer patients . Flow cytometry with the FITC-conjugated antibody can be used for potential development of clinical diagnostic assays.

What protocols maximize signal specificity when using PYGO2 antibody, FITC conjugated, in flow cytometry?

For optimal results in flow cytometry with FITC-conjugated PYGO2 antibody:

• Cell Preparation:

  • Use single-cell suspensions with viability >90%

  • For adherent cell lines, use non-enzymatic dissociation methods when possible to preserve surface epitopes

  • Include a viability dye (compatible with FITC fluorescence spectrum) to exclude dead cells

• Fixation and Permeabilization:

  • Since PYGO2 shows cytoplasmic, membranous, and nuclear localization , use a robust permeabilization protocol

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 for nuclear access

• Staining Protocol:

  • Block with 5% normal serum from the same species as secondary antibodies

  • Use recommended antibody concentration (typically 1-5 μg per million cells)

  • Include appropriate isotype control at the same concentration

  • Perform staining at 4°C for 30-45 minutes in the dark

• Signal Optimization:

  • Perform titration experiments to determine optimal antibody concentration

  • Include FMO (Fluorescence Minus One) controls to set proper gates

  • Compensate properly if using multiple fluorophores

How does PYGO2 interact with p53 pathway components, and how can antibody-based approaches elucidate this interaction?

Research has revealed important interactions between PYGO2 and the p53 pathway that can be investigated using antibody-based techniques:

• PYGO2-p53 Direct Interaction:

  • Co-immunoprecipitation experiments have confirmed interaction between Pygo2, CBP/p300, and p53

  • Using the FITC-conjugated PYGO2 antibody alongside p53 antibodies in co-localization studies can help visualize this interaction at the cellular level

• Functional Impact on p53 Activity:

  • p53 reporter assays have shown that Pygo2 deletion dampens p53 activity stimulated by nutlin-3, camptothecin, or doxorubicin

  • Chromatin immunoprecipitation (ChIP) assays using PYGO2 antibodies can determine direct binding to p53-regulated genes

• Post-translational Modifications:

  • p53 acetylation and phosphorylation, indicative of p53 activity, are more pronounced in Pygo2-expressing cells

  • Multiplexed flow cytometry with FITC-PYGO2 antibody combined with antibodies against modified p53 can quantify this relationship

• Experimental Approach:

  • Treat cells with p53 activators (nutlin-3, doxorubicin)

  • Perform parallel flow cytometry and western blotting with PYGO2 and p53 antibodies

  • Analyze correlation between PYGO2 expression and p53 activity markers

What are the technical considerations for detecting PYGO2 in different subcellular compartments?

PYGO2 exhibits localization in cytoplasmic, membranous, and nuclear compartments , presenting technical challenges for comprehensive detection:

• Fixation Optimization:

  • Cross-linking fixatives (paraformaldehyde) preserve structural relationships but may mask some epitopes

  • Alcohol-based fixatives often provide better nuclear antigen detection but may alter membrane structure

  • Sequential fixation protocols may be necessary for complete epitope preservation

• Permeabilization Strategies:

  • Nuclear localization requires more robust permeabilization (0.1-0.5% Triton X-100)

  • Membranous detection often benefits from gentler detergents (0.01-0.05% saponin)

  • Consider separate protocols optimized for each compartment if single conditions yield insufficient results

• Imaging Considerations:

  • Use confocal microscopy for accurate subcellular localization

  • Z-stack acquisition ensures complete examination of all compartments

  • Consider super-resolution techniques for detailed co-localization studies

• Quantification Approaches:

  • Implement intensity correlation analysis for co-localization studies

  • Perform subcellular fractionation followed by western blotting for biochemical validation

  • Use automated image analysis algorithms for unbiased compartment quantification

How can PYGO2 antibody be integrated into studies of immunotherapy response?

Recent research has revealed that PYGO2 plays a role in shaping the immunosuppressive tumor microenvironment, with significant implications for immunotherapy:

• Immune Checkpoint Inhibitor Response:

  • Genetic ablation or pharmacological inhibition of PYGO2 sensitizes prostate cancer to immune checkpoint blockade therapy

  • Flow cytometry with FITC-conjugated PYGO2 antibody can help stratify tumors based on expression levels for correlation with immunotherapy response

• T-cell Infiltration Assessment:

  • PYGO2 affects poor infiltration and activity of effector T cells in prostate cancer models

  • Multiplexed immunofluorescence using PYGO2 antibody alongside T-cell markers can characterize this relationship spatially within tumors

• Combination Therapy Design:

  • Research suggests a clinical path hypothesis for combining PYGO2-targeted therapy with immunotherapy in lethal prostate cancer

  • Flow cytometry panels including PYGO2 alongside immune checkpoint markers can help identify optimal combination strategies

• Myeloid-Derived Suppressor Cell Interaction:

  • PYGO2 inhibition sensitizes tumors to PMN-MDSC (polymorphonuclear myeloid-derived suppressor cells) inhibition

  • Flow cytometric analysis with the FITC-conjugated antibody can help quantify PYGO2 expression in relation to MDSC infiltration and activity

This integrated approach using PYGO2 antibody-based analyses can contribute significantly to understanding and improving immunotherapy response in multiple cancer types.

What are common issues encountered with PYGO2 antibody staining and how can they be resolved?

Researchers working with PYGO2 antibodies may encounter several technical challenges:

For the FITC-conjugated antibody specifically, remember that FITC is sensitive to high pH and photobleaching. Keep solutions slightly acidic (pH 7.2-7.4) and minimize exposure to light during all experimental steps .

How should experimental controls be designed for PYGO2 antibody validation?

Comprehensive validation requires multiple control strategies:

• Positive Controls:

  • Include known PYGO2-expressing tissues/cells (lung neuroendocrine tumors, testis)

  • Consider using cell lines with confirmed PYGO2 expression by western blot

• Negative Controls:

  • Isotype-matched control antibody at identical concentration

  • Secondary antibody-only controls (for non-conjugated primary antibodies)

  • Tissues known to express minimal PYGO2

• Technical Validation:

  • Perform peptide blocking experiments using the immunogen peptide

  • Compare staining patterns across multiple PYGO2 antibodies targeting different epitopes

  • If possible, include PYGO2 knockout/knockdown samples

• Application-Specific Controls:

  • For flow cytometry: FMO (Fluorescence Minus One) controls

  • For western blotting: molecular weight markers and loading controls

  • For IHC/ICC: tissue microarrays with gradients of expression

Proper documentation of these validation steps is essential for publication and reproducibility of research findings.

How can PYGO2 antibody, FITC conjugated be used in multiplex immunofluorescence studies?

Multiplex immunofluorescence incorporating PYGO2 detection can provide valuable insights into its interactions with other proteins and its role in different cellular contexts:

• Panel Design Considerations:

  • FITC emission spectrum (peak ~525nm) requires careful panel design to minimize spillover

  • Combine with far-red fluorophores (such as Cy5, Alexa 647) to minimize spectral overlap

  • Include markers for subcellular compartments to contextualize PYGO2 localization

• Recommended Multiplex Combinations:

  • Wnt Pathway Panel: PYGO2-FITC + β-catenin + TCF/LEF factors

  • Cell Cycle Panel: PYGO2-FITC + Ki67 + Cyclin markers

  • EMT Panel: PYGO2-FITC + E-cadherin + Vimentin

  • Tumor Microenvironment Panel: PYGO2-FITC + CD8 + CD4 + FOXP3

• Technical Optimization:

  • Sequential staining may be necessary to prevent antibody cross-reactivity

  • Consider signal amplification methods for weaker markers

  • Implement spectral unmixing for highly multiplexed panels

• Analysis Approaches:

  • Quantify co-localization coefficients between PYGO2 and interacting proteins

  • Perform neighborhood analysis in tissue sections to identify spatial relationships

  • Consider machine learning approaches for pattern recognition in complex datasets

What role does PYGO2 play in cancer stem cell biology and how can this be studied?

PYGO2 regulates stem cell self-renewal and somatic cell division , making it particularly relevant for cancer stem cell research:

• Experimental Approaches:

  • Flow cytometry with FITC-conjugated PYGO2 antibody alongside established cancer stem cell markers

  • Sphere formation assays comparing PYGO2-high versus PYGO2-low sorted populations

  • Lineage tracing experiments in model systems with PYGO2 manipulation

• Mechanistic Studies:

  • ChIP-seq to identify PYGO2 binding sites at stem cell-related gene promoters

  • RNA-seq of PYGO2-manipulated cells to identify transcriptional networks

  • Protein-protein interaction studies focusing on chromatin modifiers

• Clinical Correlation:

  • Analysis of PYGO2 expression in treatment-resistant tumor populations

  • Correlation of PYGO2 levels with cancer recurrence and metastasis

  • Development of therapeutic strategies targeting PYGO2 in cancer stem cells

This research direction could provide valuable insights into the fundamental mechanisms of tumor initiation, progression, and therapy resistance.

How might PYGO2 antibodies contribute to developing targeted cancer therapies?

Research suggests multiple promising avenues for therapeutic development:

• Combination with Immunotherapy:

  • PYGO2 inhibition sensitizes tumors to immune checkpoint blockade, adoptive T-cell therapy, and PMN-MDSC inhibition

  • PYGO2 antibodies can help identify patients most likely to benefit from such combination approaches

• Biomarker Development:

  • Abnormal PYGO2 expression correlates with poor prognosis in several cancer types

  • Flow cytometry with standardized protocols could yield clinically applicable prognostic tests

• Target Validation:

  • PYGO2 antibodies can validate the specificity of newly developed small molecule inhibitors

  • Pharmacodynamic studies using the antibody can confirm target engagement in vivo

• Delivery System Development:

  • Antibody-drug conjugates targeting PYGO2 could be explored

  • Nanoparticle targeting strategies using PYGO2-specific binding domains