CPNE8 Antibody

What is CPNE8 and why is it significant in research?

CPNE8, or Copine VIII, is a member of the copine family, which are calcium-dependent membrane-binding proteins. The significance of CPNE8 in research has grown substantially due to its identified roles in several malignancies. Research has demonstrated that CPNE8 exhibits oncogenic properties in various cancers including gastric cancer (STAD), head and neck squamous cell carcinoma (HNSC), and esophageal carcinoma (ESCA) . Its calcium-dependent membrane binding properties suggest involvement in cellular signaling pathways, making it an important target for investigating cancer mechanisms and potential therapeutic approaches .

What applications are CPNE8 antibodies typically used for?

CPNE8 antibodies are primarily utilized in several fundamental research applications:

• Immunohistochemistry (IHC): For detecting CPNE8 in tissue sections, which is particularly valuable for analyzing expression patterns in tumor samples versus normal tissues

• Western Blot (WB): For protein expression and quantification studies of CPNE8 in cell and tissue lysates

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative determination of CPNE8 in various sample types

The diverse application range allows researchers to examine CPNE8 expression, localization, and functions across different experimental systems and disease models, facilitating comprehensive investigations into its biological roles .

What species reactivity should I consider when selecting a CPNE8 antibody?

When selecting a CPNE8 antibody, species reactivity is a critical consideration to ensure experimental validity. Based on available antibodies, the following species reactivity profiles are observed:

• Human-specific CPNE8 antibodies: These are the most commonly available and have been extensively validated for human samples

• Multi-species reactive antibodies: Several antibodies demonstrate cross-reactivity with mouse and rat CPNE8

• Broader reactivity spectrum: Some antibodies show extended cross-reactivity with additional species including cow, guinea pig, rabbit, and zebrafish (Danio rerio)

For comparative studies across species, selecting antibodies with validated cross-reactivity is essential. If your research focuses exclusively on human samples, human-specific antibodies may provide optimal specificity. Always review validation data for your specific application and species of interest to ensure reliable results .

How should I validate a CPNE8 antibody before use in my experiments?

Proper antibody validation is crucial for ensuring experimental reliability. For CPNE8 antibodies, implement the following validation workflow:

• Positive and negative controls: Use cell lines or tissues with known CPNE8 expression levels. For negative controls, consider using CPNE8 knockdown cells or tissues where CPNE8 is naturally not expressed.

• Verification across multiple applications: If planning to use the antibody for multiple applications (IHC, WB, ELISA), verify specificity in each application independently .

• Epitope consideration: Check if the antibody recognizes specific domains (N-terminal vs. AA 349-498) to ensure it will detect your protein of interest, especially if studying specific isoforms or truncated variants .

• Cross-reactivity assessment: If working with non-human samples, validate the cross-reactivity claims from manufacturers with your specific samples .

• Dilution optimization: Test several dilutions around the manufacturer's recommended range (e.g., 1:50-1:1000 for IHC) to determine optimal signal-to-noise ratio for your specific experimental conditions .

This systematic validation approach will significantly enhance the reliability and reproducibility of your CPNE8-related research findings.

How can CPNE8 antibodies be utilized in cancer research?

CPNE8 antibodies serve as valuable tools in cancer research through multiple sophisticated applications:

These applications demonstrate how CPNE8 antibodies can be integrated into translational cancer research pipelines to advance understanding of cancer biology and improve patient stratification strategies.

What methodological considerations are important when using CPNE8 antibodies for immunohistochemistry?

When using CPNE8 antibodies for immunohistochemistry (IHC), several critical methodological considerations can optimize results:

• Fixation protocol optimization: Standardize fixation times (typically 24-48 hours in 10% neutral buffered formalin) to maintain epitope integrity. Overfixation can mask epitopes, while underfixation may compromise tissue morphology.

• Antigen retrieval method selection: For CPNE8 antibodies, heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is commonly effective. Compare both methods to determine optimal retrieval for your specific antibody and tissue type .

• Antibody dilution and incubation parameters: The recommended dilution range for CPNE8 antibodies in IHC applications typically falls between 1:50-1:1000 . Optimize by testing multiple dilutions within this range.

• Detection system considerations: For weakly expressed CPNE8, consider using amplification systems like polymer-based detection or tyramide signal amplification to enhance sensitivity without increasing background.

• Counterstaining protocol adaptation: When evaluating CPNE8 subcellular localization, adjust hematoxylin counterstaining intensity to provide sufficient nuclear contrast without obscuring antibody signal.

• Multi-antigen staining approach: For co-localization studies with other proteins in the focal adhesion pathway, implement sequential or multiplex IHC protocols with carefully selected antibody pairs that avoid cross-reactivity.

• Quantification methodology standardization: Establish consistent scoring systems for CPNE8 expression in tissues, considering both staining intensity and percentage of positive cells to create H-scores or other semi-quantitative metrics.

These methodological refinements can significantly enhance the reliability and interpretability of CPNE8 IHC results in research applications.

How does CPNE8 expression correlate with tumor immune microenvironment?

CPNE8 expression demonstrates significant associations with the tumor immune microenvironment across multiple cancer types, with particularly well-characterized relationships in ESCA, STAD, and THCA:

• Stromal and immune cell infiltration correlation: In STAD, CPNE8 expression shows positive correlation with stromal score (R=0.38, P=1.8e-14), indicating association with non-tumor cells in the microenvironment .

• Dendritic cell association: CPNE8 expression positively correlates with dendritic cell infiltration in ESCA (R=0.38, P=3.4e-06), suggesting potential involvement in antigen presentation and T cell activation processes .

• Regulatory T cell inverse relationship: In ESCA, CPNE8 expression negatively correlates with regulatory T cell infiltration (R=-0.41, P=3.2e-07), potentially indicating an immunosuppressive role .

• Natural killer cell subset differential association: Interestingly, CPNE8 demonstrates opposing relationships with different NK cell subsets in ESCA - positive correlation with CD56bright NK cells (R=0.327, P=6.47e-06) but negative correlation with CD56dim NK cells (R=-0.314, P=1.45e-05) .

• Myeloid cell correlations: CPNE8 expression negatively correlates with monocyte abundance in ESCA (R=-0.376, P=1.72e-07) but positively with macrophage abundance in STAD (R=0.322, P=2.33e-11) .

These relationships suggest CPNE8 may function as an immunomodulatory factor across cancers, potentially influencing both innate and adaptive immune responses in the tumor microenvironment. When designing studies to investigate these associations, implementing multiplex immunofluorescence or spatial transcriptomics approaches alongside CPNE8 antibody staining can provide higher-resolution insights into these complex cellular interactions.

What is the relationship between CPNE8 and focal adhesion in cancer progression?

Research has established that CPNE8 promotes cancer progression through regulation of focal adhesion pathways, particularly in gastric cancer:

• Mechanistic relationship: CPNE8 has been demonstrated to promote gastric cancer metastasis specifically through modulation of focal adhesion complexes. This relationship is functionally significant, as the metastasis-promoting effects of CPNE8 can be reversed by focal adhesion kinase (FAK) inhibition using compounds such as GSK2256098 or by direct knockdown of FAK .

• Experimental approach for investigation: To study this relationship, researchers can implement several techniques:

  • Co-immunoprecipitation with CPNE8 antibodies to identify binding partners within focal adhesion complexes

  • Phospho-specific antibodies to monitor FAK activation status in CPNE8-overexpressing or knockdown models

  • Immunofluorescence microscopy to visualize colocalization of CPNE8 with focal adhesion components such as paxillin, vinculin, and phospho-FAK

  • Cell migration and invasion assays combined with FAK inhibitors to assess functional consequences

• Translational implications: The connection between CPNE8 and focal adhesion suggests that patients with high CPNE8 expression might benefit from therapeutic approaches targeting focal adhesion pathways. Implementing CPNE8 antibody-based screening could potentially identify patients suitable for FAK inhibitor therapies .

This relationship highlights how CPNE8 connects to established cancer progression mechanisms and provides rationale for targeting focal adhesion in CPNE8-overexpressing tumors.

How can CPNE8 antibodies be utilized in gene set enrichment analysis (GSEA) validation?

CPNE8 antibodies can serve as powerful validation tools for gene set enrichment analysis (GSEA) findings through a multi-layered experimental approach:

• Pathway validation strategy: GSEA of CPNE8-correlated genes has identified enrichment in several key pathways including antigen processing and presentation, Toll-like receptor signaling, calcium signaling, and cell adhesion molecules . To validate these bioinformatic findings:

  • Use CPNE8 antibodies alongside antibodies for key proteins in these pathways in co-immunoprecipitation experiments to confirm physical interactions

  • Implement proximity ligation assays to visualize and quantify protein-protein interactions in situ

  • Conduct ChIP-seq experiments in CPNE8-manipulated systems to identify direct transcriptional effects

• Functional validation approach: For validating immune-related pathway connections:

  • Perform CPNE8 overexpression or knockdown in relevant cell models, followed by flow cytometry analysis of immune checkpoint molecules

  • Measure cytokine production changes in response to CPNE8 manipulation, correlating with antibody-based detection of pathway activation

  • Implement in vitro co-culture systems with immune cells to assess functional consequences of CPNE8 expression

• Translational validation methodology: For clinical relevance:

  • Develop multiplex IHC panels including CPNE8 antibodies and markers for enriched pathways to analyze patient samples

  • Correlate CPNE8 protein expression with pathway activity scores from transcriptomic data

  • Create predictive models incorporating CPNE8 protein levels and pathway signatures

This integrated approach transforms computational GSEA findings into experimentally validated mechanisms, strengthening the biological understanding of CPNE8's role in cancer progression and immune modulation.

What factors affect CPNE8 antibody sensitivity and specificity?

Several critical factors influence the sensitivity and specificity of CPNE8 antibodies:

Understanding and optimizing these factors will significantly enhance the reliability of CPNE8 antibody-based experiments and reduce troubleshooting time.

How should I optimize Western blot protocols for CPNE8 detection?

Optimizing Western blot protocols for CPNE8 detection requires systematic adjustment of several parameters:

• Sample preparation refinement:

  • Use RIPA buffer supplemented with both protease inhibitors and phosphatase inhibitors

  • For membrane-associated CPNE8 fractions, consider membrane protein extraction kits

  • Standardize protein quantification and loading (25-50μg total protein typically provides adequate signal)

• Electrophoresis and transfer optimization:

  • CPNE8 has a molecular weight of approximately 62 kDa; use 10% acrylamide gels for optimal resolution

  • Extend transfer time to 2 hours or implement overnight transfer at lower voltage for complete transfer of CPNE8

  • Consider wet transfer systems over semi-dry for more consistent transfer of CPNE8

• Blocking strategy selection:

  • Compare 5% non-fat milk versus 5% BSA in TBST for optimal signal-to-noise ratio

  • Extended blocking (2 hours at room temperature or overnight at 4°C) may reduce background

• Antibody dilution optimization:

  • Test multiple dilutions within manufacturer's recommended range (typically 1:500-1:2000)

  • Extend primary antibody incubation to overnight at 4°C to enhance sensitivity

  • For secondary antibodies, 1:5000-1:10000 dilutions typically provide good results

• Detection system selection:

  • For low abundance CPNE8, enhanced chemiluminescence (ECL) plus or super-signal systems provide greater sensitivity

  • Consider using fluorescently-labeled secondary antibodies for multiplex detection and more precise quantification

• Confirmation strategies:

  • Verify specificity using positive control lysates from cells with confirmed CPNE8 expression

  • Include CPNE8 knockdown or knockout samples as negative controls

  • For additional validation, consider using two different CPNE8 antibodies targeting distinct epitopes

This optimization strategy will help establish a robust and reproducible Western blot protocol for CPNE8 detection across diverse experimental conditions.

What controls should be included when using CPNE8 antibodies for experimental validation?

Implementing comprehensive controls is essential for ensuring the validity of CPNE8 antibody-based experiments:

• Positive controls:

  • Cell lines with confirmed high CPNE8 expression (based on literature, cancer cell lines from STAD, HNSC or ESCA may be appropriate)

  • Recombinant CPNE8 protein for Western blot or ELISA applications

  • Tissues with known CPNE8 expression based on published immunohistochemistry data

• Negative controls:

  • CPNE8 knockdown or knockout cell lines generated via siRNA, shRNA, or CRISPR-Cas9

  • Cell lines naturally expressing minimal CPNE8

  • Primary antibody omission controls for all immunostaining procedures

  • Isotype controls using non-specific IgG from the same host species at equivalent concentrations

• Specificity controls:

  • Peptide competition assays using the immunizing peptide to confirm antibody specificity

  • Comparison of staining patterns using multiple CPNE8 antibodies targeting different epitopes

  • Correlation of protein detection with mRNA expression data from the same samples

• Method-specific controls:

  • For IHC: Tissue microarrays containing multiple cancer and normal tissue types to verify staining patterns across diverse samples

  • For Western blot: Molecular weight markers and loading controls (β-actin, GAPDH, or total protein staining)

  • For immunoprecipitation: Input, flow-through, and non-specific binding controls

  • For immunofluorescence: Autofluorescence controls and single-stain controls for co-localization studies

• Biological validation controls:

  • CPNE8 overexpression systems to confirm antibody detection capability across expression levels

  • Treatment conditions known to alter CPNE8 expression or localization based on literature

  • Parallel analysis using complementary detection methods (e.g., RNA-seq, proteomics)

Implementing these comprehensive controls will substantially strengthen the validity and interpretability of CPNE8 antibody-based research findings.

How might CPNE8 antibodies be utilized in investigating immunotherapy response biomarkers?

CPNE8 antibodies present promising opportunities for immunotherapy response biomarker research, building on established correlations between CPNE8 and immune parameters:

• Predictive biomarker development strategy: Given the significant correlations between CPNE8 expression and tumor mutation burden (TMB) in HNSC (R=0.265, P=2.23e-09), THYM (R=0.331, P=0.0002), and other cancers , CPNE8 immunohistochemistry could be integrated into multiplex IHC panels alongside established predictive markers like PD-L1. This approach could potentially enhance prediction accuracy for immunotherapy response.

• Immune cell interaction assessment: CPNE8's correlations with diverse immune cell populations suggest it may influence the tumor immune microenvironment . Researchers could implement:

  • Multiplex immunofluorescence panels combining CPNE8 antibodies with immune checkpoint markers

  • Spatial analysis of CPNE8-expressing tumor cells relative to TIL distribution

  • Longitudinal analysis of CPNE8 expression before and during immunotherapy treatment

• Mechanistic investigation approach: To understand how CPNE8 might modulate immunotherapy response:

  • Evaluate how CPNE8 knockdown/overexpression affects PD-L1 expression and T cell co-culture killing assays

  • Assess CPNE8's impact on antigen presentation pathway components identified in GSEA analyses

  • Investigate how CPNE8-modulated focal adhesion signaling might intersect with immune cell recruitment and activation

• Clinical correlation methodology: For translational validation:

  • Develop standardized CPNE8 IHC scoring systems for prospective clinical trials

  • Create tissue microarrays from immunotherapy-treated patient cohorts with known response data

  • Correlate CPNE8 expression patterns with immune-related adverse events and response durability

This research direction could potentially identify CPNE8 as a novel component of the complex biomarker landscape for cancer immunotherapy response prediction.

What potential exists for developing CPNE8-targeted therapeutic approaches?

The emerging understanding of CPNE8's role in cancer progression suggests several promising avenues for therapeutic development:

• Focal adhesion pathway intervention: Research demonstrating that CPNE8 promotes cancer metastasis through focal adhesion pathways provides a clear rationale for therapeutic targeting . Potential approaches include:

  • Combination therapy integrating CPNE8 status assessment with existing FAK inhibitors like GSK2256098

  • Development of small molecules specifically disrupting CPNE8-FAK interactions

  • Peptide-based inhibitors designed to interfere with CPNE8 binding to focal adhesion components

• Immune microenvironment modulation: CPNE8's correlations with immune cell infiltration suggest potential immunotherapeutic applications:

  • Exploring how CPNE8 inhibition might enhance immunotherapy response in cancers with high CPNE8 expression

  • Investigating whether CPNE8 status can guide selection of specific immunotherapy combinations

  • Developing strategies to reverse CPNE8-mediated immunosuppressive mechanisms

• RNA interference approaches: Given CPNE8's oncogenic properties in several cancers , direct targeting via RNAi holds promise:

  • siRNA delivery systems specifically targeting CPNE8-overexpressing tumor cells

  • Exploration of CPNE8 regulation by identified microRNAs such as miR-222-3p

  • Long non-coding RNA modulation strategies targeting PWAR5 and LIFR-AS1, which regulate CPNE8 expression

• Antibody-based therapeutic strategies:

  • Antibody-drug conjugates utilizing CPNE8 antibodies for targeted delivery of cytotoxic payloads

  • Development of function-blocking antibodies that inhibit CPNE8's interaction with focal adhesion components

  • Bispecific antibodies linking CPNE8-expressing tumor cells with immune effector cells

These therapeutic directions would benefit from further mechanistic understanding of CPNE8's roles across cancer types and careful patient stratification based on CPNE8 expression patterns.

How might multi-omics approaches incorporate CPNE8 antibody-based data for comprehensive cancer characterization?

Integrating CPNE8 antibody-based data into multi-omics cancer characterization offers powerful opportunities for comprehensive disease understanding:

• Proteogenomic integration framework: Combining CPNE8 protein detection with genomic alterations creates a more complete biological picture:

  • Correlate CPNE8 protein expression (detected via antibody-based methods) with copy number variations and mutations

  • Integrate CPNE8 protein localization data with transcriptomic profiles

  • Analyze post-translational modifications of CPNE8 in relation to pathway activation

• Spatial multi-omics approach: Mapping CPNE8 expression in spatial context can reveal important tumor heterogeneity:

  • Implement multiplex IHC with CPNE8 antibodies alongside markers for immune cells, stromal components, and signaling pathways

  • Combine with spatial transcriptomics from matched regions to correlate protein expression with gene signatures

  • Develop computational frameworks that integrate spatial CPNE8 protein data with other spatially resolved datasets

• Functional multi-omics integration: Connecting CPNE8 expression with functional outcomes:

  • Correlate CPNE8 antibody-based detection with phosphoproteomics data focusing on focal adhesion pathways

  • Integrate with metabolomics to identify metabolic signatures associated with CPNE8-high versus CPNE8-low tumors

  • Link CPNE8 protein levels with drug sensitivity profiles across patient-derived models

• Clinical multi-omics implementation: Translating integrated data to clinical applications:

  • Develop machine learning algorithms incorporating CPNE8 protein expression alongside genomic and clinical data for outcome prediction

  • Create integrated biomarker panels combining CPNE8 IHC with other molecular features for patient stratification

  • Design treatment selection workflows that consider CPNE8 status in the context of comprehensive molecular profiling

This multi-omics framework transforms CPNE8 antibody data from isolated measurements to components of an integrated biological understanding, potentially accelerating precision oncology applications.