CYP2J2 Antibody

What is CYP2J2 and why is it important in cardiovascular research?

CYP2J2 is a member of the Cytochrome P450 family of enzymes and serves as the most abundant epoxygenase in the heart. Its significance lies in its ability to bioactivate arachidonic acid to epoxyeicosatrienoic acids (EETs), which play crucial roles in cardiovascular health. Proteomic studies have revealed that CYP2J2 protein levels are significantly lower in cardiac tissue from patients with cardiomyopathy compared to healthy controls . The enzyme regulates diverse transcriptional programs in cardiomyocytes, including those involved in ion channel signaling, development, extracellular matrix organization, and metabolism . Research has demonstrated that CYP2J2 overexpression can protect against abdominal aortic aneurysm (AAA) development in animal models, highlighting its potential as a therapeutic target in cardiovascular disease .

What are the recommended applications for CYP2J2 antibodies in cardiac research?

CYP2J2 antibodies can be employed in multiple experimental applications with specific recommendations for each:

For immunohistochemical applications, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 can serve as an alternative . When selecting application-specific conditions, it's advisable to perform preliminary optimization experiments to determine the optimal antibody concentration for each specific tissue or cell type.

How should I validate CYP2J2 antibody specificity in my experimental system?

Validating antibody specificity is essential for generating reliable data. A multi-faceted approach is recommended:

• Genetic Validation: Utilize siRNA-mediated knockdown of CYP2J2 as demonstrated in published research. A properly specific antibody should show significantly reduced signal in Western blot and immunostaining following knockdown .

• Functional Correlation: Measure CYP2J2 enzymatic activity using terfenadine as a probe substrate. Research has shown that siRNA treatment reduces both CYP2J2 protein levels (detected by antibody) and terfenadine metabolism by >90%, confirming antibody specificity .

• Tissue Distribution Analysis: Compare staining patterns across tissues with known differential CYP2J2 expression. The expected pattern includes strong signal in heart tissue, with detectable expression in liver tissue .

• Molecular Weight Verification: CYP2J2 should be detected at 58-65 kDa in Western blot applications . Significant deviation from this range may indicate non-specific binding.

• Multiple Antibody Validation: When possible, use antibodies recognizing different epitopes of CYP2J2 and verify consistent results across antibodies.

How can I design experiments to investigate the relationship between CYP2J2 expression and EET production?

Investigating this relationship requires a parallel quantification strategy:

Experimental Design:

• CYP2J2 Manipulation Models:

  • Viral vector-mediated overexpression (rAAV-CYP2J2 as described in the literature)

  • siRNA-mediated knockdown

  • Pharmacological inhibition (e.g., danazol)

• Quantification Methods:

  • Western blot analysis of CYP2J2 protein using validated antibodies

  • LC-MS/MS measurement of 11,12-EET and 14,15-EET (primary CYP2J2 products)

  • Measure corresponding DHETs to account for EET metabolism

Sample Data Table from Published Research:

When analyzing data, calculate the correlation coefficient between CYP2J2 protein levels and EET concentrations to establish a quantitative relationship . This experimental approach provides strong evidence for the functional consequences of altered CYP2J2 expression.

What methodological approaches should I use when studying CYP2J2 in cardiac disease models?

For robust experimental design in cardiac disease models:

In Human Studies:

• Compare CYP2J2 expression in diseased versus healthy cardiac tissue using proteomic mass spectrometry and validate with antibody-based methods

• Correlate expression levels with clinical parameters and disease severity

In Cellular Models:

• Culture human adult ventricular cardiomyocytes

• Perform targeted silencing of CYP2J2 using siRNA as demonstrated in published research

• Assess transcriptional responses using RNA-seq

• Measure protein expression changes via Western blot with validated CYP2J2 antibodies

• Evaluate enzymatic activity using terfenadine metabolism assays

In Animal Models:

• Utilize established cardiac disease models (e.g., pressure overload, Ang II-induced AAA)

• Deliver rAAV-CYP2J2 for overexpression studies

• Assess cardiac function (echocardiography)

• Analyze tissue remodeling (histology, immunostaining)

• Measure EET production via LC-MS/MS

The research demonstrates that CYP2J2 knockdown elicits widespread alterations in gene expression of ventricular cardiomyocytes, affecting ion channel signaling, development, extracellular matrix, and metabolism . Conversely, CYP2J2 overexpression provides protection against AAA development by attenuating matrix metalloproteinase expression and activity .

How do I optimize CYP2J2 antibody staining in cardiac tissue sections?

For consistent and specific CYP2J2 immunostaining in cardiac tissue:

Tissue Preparation:

• Fix tissues in 10% neutral buffered formalin (optimal: 24 hours for cardiac tissue)

• Process and embed in paraffin

• Section at 4-6 μm thickness

Antigen Retrieval (Critical Step):

• Primary method: Heat-induced epitope retrieval using TE buffer pH 9.0

• Alternative: Citrate buffer pH 6.0 if primary method yields unsatisfactory results

Staining Protocol Optimization:

• Perform antibody titration experiments (1:50, 1:100, 1:200, 1:500) to determine optimal dilution

• Test multiple incubation conditions (overnight at 4°C vs. 1-2 hours at room temperature)

• Compare different detection systems (HRP-DAB vs. fluorescent)

• Include positive controls (human liver tissue, mouse heart tissue)

• Include negative controls (omit primary antibody or use isotype control)

Troubleshooting Common Issues:

• Weak signal: Increase antibody concentration, extend incubation time, or enhance antigen retrieval

• High background: Increase blocking time, use more stringent washing, or reduce antibody concentration

• Patchy staining: Ensure uniform section thickness and complete deparaffinization

How can I effectively use CYP2J2 antibodies in siRNA knockdown validation experiments?

Validating CYP2J2 siRNA knockdown requires careful experimental design:

Experimental Setup:

• Treat cells with CYP2J2-specific siRNA and scramble siRNA control

• Harvest cells at multiple time points (24, 48, 72 hours post-transfection)

• Divide samples for parallel analysis:

  • Protein expression (Western blot)

  • Enzymatic activity (terfenadine metabolism)

  • mRNA levels (qRT-PCR)

Western Blot Protocol:

• Load normalized protein amounts (20-50 μg per lane)

• Use gradient gels (8-12%) for optimal resolution around 58-65 kDa

• Incubate with CYP2J2 antibody at optimized dilution (1:1000-1:4000 recommended)

• Include loading control (β-actin, GAPDH)

Validation Metrics:

• Calculate percent knockdown relative to scramble siRNA control

• Confirm that protein reduction correlates with decreased enzymatic activity

• Research has shown that effective knockdown results in >90% decrease in terfenadine metabolism

Documentation Example:

This comprehensive validation approach ensures that observed phenotypic changes can be reliably attributed to specific CYP2J2 knockdown.

How should I interpret changes in CYP2J2 expression levels in different cardiac pathologies?

Interpreting CYP2J2 expression changes requires consideration of disease context and mechanistic implications:

Disease-Specific Patterns:

• Cardiomyopathy: Research demonstrates significantly reduced CYP2J2 protein levels in patients with non-ischemic cardiomyopathy compared to controls . This suggests potential loss of cardioprotective mechanisms.

• Heart Failure: Evaluate whether expression changes correlate with disease progression and functional parameters.

• Arrhythmias: CYP2J2 silencing affects ion channel gene expression, potentially impacting cardiac electrophysiology .

Mechanistic Considerations:

• Determine whether CYP2J2 changes precede or follow pathological alterations

• Assess corresponding changes in EET levels to establish functional consequences

• Consider activation of downstream signaling pathways (e.g., PPARγ activation, which mediates CYP2J2's protective effects against AAA )

Analytical Framework:

• Quantify CYP2J2 expression using calibrated antibody-based methods

• Normalize to appropriate reference (loading controls for Western blot, housekeeping proteins for IHC)

• Correlate with clinical/functional parameters

• Consider confounding factors (medications, comorbidities)

This analytical approach enables distinction between pathophysiologically significant changes and secondary adaptations.

How do I address potential cross-reactivity with other CYP family members in CYP2J2 antibody experiments?

Cross-reactivity with related CYP enzymes presents a significant challenge for CYP2J2 antibody specificity. A systematic approach includes:

Pre-Experimental Assessment:

• Review antibody validation data for known cross-reactivities

• Examine the immunogen sequence for homology with other CYP enzymes

Experimental Validation:

• Genetic Controls: siRNA knockdown of CYP2J2 should significantly reduce antibody signal if specific. Research demonstrates >90% reduction in CYP2J2 activity following siRNA treatment .

• Functional Discrimination: Combine antibody detection with CYP2J2-specific activity assays (terfenadine metabolism or EET production).

• Expression Pattern Analysis: Compare antibody staining patterns with known tissue distribution of CYP2J2 versus other CYP enzymes. CYP2J2 antibodies should show strong reactivity in heart tissue, which is where CYP2J2 is most abundant .

• Molecular Weight Verification: CYP2J2 has an observed molecular weight of 58-65 kDa . Carefully analyze Western blot bands to ensure they appear at the correct size.

• Multiple Antibody Validation: Use antibodies targeting different epitopes of CYP2J2 to confirm consistent results.

Documentation Best Practices:

• Report all specificity testing methods in publications

• Acknowledge any known cross-reactivities

• Document controls used to ensure specificity

This comprehensive approach minimizes false-positive results due to cross-reactivity with other CYP family members.

How can CYP2J2 antibodies be used to study the relationship between CYP2J2 and cardiac ion channels?

Given that CYP2J2 silencing affects ion channel gene expression in cardiomyocytes, studying this relationship requires specialized approaches:

Co-localization Studies:

• Perform multi-label immunofluorescence with CYP2J2 antibody and antibodies against specific ion channels

• Use confocal microscopy with sequential scanning to minimize bleed-through

• Calculate co-localization metrics (Pearson's correlation coefficient)

Functional Correlation Studies:

• Correlate CYP2J2 expression (quantified by antibody-based methods) with electrophysiological parameters

• Measure ion channel current densities in cells with varied CYP2J2 expression levels

• Research has shown that CYP2J2 silencing affects the expression of several members of ion channel modules that have well-known pathogenetic roles in cardiac dysrhythmias

Experimental Model System:

• Human adult ventricular cardiomyocytes provide an ideal system for studying this relationship

• Modulate CYP2J2 expression using siRNA techniques or overexpression vectors

• Perform comprehensive transcriptional profiling to identify affected ion channel genes

This integrated approach can elucidate how CYP2J2 influences cardiac electrophysiology and potentially identify new therapeutic targets for cardiac arrhythmias.

What role might CYP2J2 play in personalized medicine approaches for cardiac diseases?

CYP2J2's differential expression in cardiac pathologies suggests potential applications in personalized medicine:

Biomarker Development:

• CYP2J2 protein levels are significantly lower in cardiac tissue from patients with cardiomyopathy

• Quantitative immunohistochemistry using validated CYP2J2 antibodies could identify patients with altered CYP2J2 expression

Therapeutic Targeting:

• Research demonstrates that rAAV-mediated CYP2J2 overexpression protects against AAA development

• Patient stratification based on CYP2J2 expression levels might identify those most likely to benefit from EET-based therapies

Genetic Considerations:

• CYP2J2 polymorphisms could affect enzyme activity and expression

• Antibody-based methods can verify the impact of genetic variants on protein expression

Monitoring Treatment Response:

• Serial measurement of CYP2J2 expression and activity might help track therapeutic efficacy

• Changes in CYP2J2-regulated pathways could serve as surrogate markers for treatment response

As our understanding of CYP2J2's role in cardiovascular health evolves, antibody-based detection methods will remain essential tools for translating basic research findings into clinical applications.