CYP19-4 Antibody

What is CYP19 and what is its biological significance?

CYP19, commonly known as aromatase, is an enzyme responsible for catalyzing the conversion of androgens to estrogens. It represents a critical component in steroid hormone biosynthesis and plays essential roles in reproductive development, fertility, bone health, and various pathological conditions. The enzyme is encoded by the CYP19 gene, which can have multiple promoters driving its expression in different tissues. Aromatase activity has been demonstrated to be markedly elevated in certain disease conditions, particularly in hepatocellular carcinoma (HCC) . The enzyme's function is highly conserved across species, highlighting its fundamental biological importance in vertebrate evolution .

How does CYP19-4 antibody differ from other CYP19 antibodies?

CYP19-4 antibody is specifically designed to target unique epitopes in the CYP19 protein structure, offering improved specificity for particular research applications. When selecting an antibody for aromatase research, it's essential to consider the specific isoform or domain being targeted. In evolutionary studies, researchers have identified multiple CYP19-like proteins in primitive chordates such as amphioxus, with distinct expression patterns and potentially different functions . For immunohistochemistry or Western blotting, CYP19-4 antibody provides superior sensitivity for detecting low-level expression in tissues where CYP19 is not abundantly expressed.

What are the most common applications for CYP19-4 antibody in basic research?

CYP19-4 antibody is primarily utilized in:

• Immunohistochemistry (IHC) to localize aromatase expression in tissue sections

• Western blotting for quantitative protein expression analysis

• Immunoprecipitation studies to examine protein-protein interactions

• Flow cytometry for cellular-level expression analysis

• Chromatin immunoprecipitation (ChIP) to study transcriptional regulation

For in situ hybridization studies, researchers have successfully used specific probes to detect cyp19-like gene expression in different tissues, which can be complemented with antibody-based protein detection for comprehensive analysis . Gene expression studies frequently employ both real-time PCR and antibody-based methods to correlate transcript and protein levels.

What are the optimal protocols for using CYP19-4 antibody in Western blotting?

For optimal Western blotting results with CYP19-4 antibody:

• Sample preparation: Extract proteins using RIPA buffer supplemented with protease inhibitors to prevent degradation.

• Protein loading: Load 20-50 μg of total protein per lane for cell lysates or tissue homogenates.

• Separation: Use 10% SDS-PAGE gels for optimal separation of the ~55 kDa aromatase protein.

• Transfer: Employ semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 1 hour.

• Blocking: Block membranes with 5% non-fat milk in TBST for 1 hour at room temperature.

• Primary antibody: Dilute CYP19-4 antibody at 1:1000 in blocking buffer and incubate overnight at 4°C.

• Secondary antibody: Use HRP-conjugated anti-species antibody at 1:5000 dilution for 1 hour at room temperature.

• Detection: Develop using ECL substrate with exposure times optimized for signal intensity.

For tissues with low aromatase expression, signal amplification methods may be necessary. When analyzing expression differences between experimental groups, normalization to housekeeping proteins such as β-actin or GAPDH is essential for accurate quantification .

How should CYP19-4 antibody be utilized for immunohistochemistry studies?

For immunohistochemistry applications:

• Tissue fixation: Fix tissues in 4% paraformaldehyde for 24 hours before paraffin embedding.

• Sectioning: Prepare 5-7 μm sections on positively charged slides.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes.

• Blocking: Block endogenous peroxidase with 3% H₂O₂ and non-specific binding with 5-10% normal serum.

• Primary antibody: Apply CYP19-4 antibody at 1:100-1:200 dilution and incubate overnight at 4°C.

• Detection system: Use biotin-streptavidin HRP detection or polymer-based detection systems.

• Visualization: Develop with DAB substrate and counterstain with hematoxylin.

• Controls: Always include positive and negative controls to validate staining specificity.

This protocol has been effective for detecting aromatase expression in various tissues, including liver samples from hepatocellular carcinoma patients and gonadal tissues in evolutionary studies . For dual immunofluorescence studies, appropriate fluorophore-conjugated secondary antibodies should be selected based on the primary antibody host species.

What is the recommended approach for generating and validating polyclonal antibodies against CYP19?

For generating polyclonal antibodies against CYP19:

• Peptide design: Select unique, antigenic regions of the CYP19 protein (typically 15-20 amino acids), such as the sequences "N′-CREELKTAPPSDKPD-C′" and "N′-CPSRDHKSLDVSRNL-C′" that have been successfully used for Cyp19-like proteins .

• Conjugation: Conjugate the peptide to a carrier protein (KLH or BSA) to enhance immunogenicity.

• Immunization: Use 6-week-old female mice or rabbits for immunization following standard protocols with 3-4 booster injections.

• Antibody harvest: Collect serum and purify IgG fraction using protein A/G columns.

• Validation steps:

  • ELISA against the immunizing peptide

  • Western blot against recombinant protein and native samples

  • Immunoprecipitation followed by mass spectrometry

  • Immunohistochemistry with appropriate positive and negative controls

  • Testing on knockout/knockdown samples when available

Special attention should be paid to validation across different experimental conditions and species, particularly when studying evolutionarily conserved proteins like aromatase .

How can CYP19-4 antibody be applied in studying the role of aromatase in hepatocellular carcinoma?

CYP19-4 antibody provides valuable tools for investigating aromatase's role in hepatocellular carcinoma through several approaches:

• Expression profiling: Compare aromatase protein levels between HCC and adjacent non-tumor tissues using immunohistochemistry and Western blotting to detect potential upregulation, as suggested by studies showing elevated hepatic aromatase activity in HCC .

• Correlation with clinicopathological features: Analyze aromatase expression in relation to tumor grade, stage, and patient outcomes through tissue microarray analysis.

• Mechanistic studies: Combine with other markers to investigate the relationship between aromatase expression and estrogen receptor signaling in HCC progression.

• Genotype-phenotype correlation: Investigate how CYP19 promoter polymorphisms (such as the rs10459592 A/C variation) affect protein expression levels and correlate with HCC risk .

• Therapeutic target assessment: Evaluate aromatase as a potential target in HCC by examining expression changes in response to experimental treatments.

Research has demonstrated that transcriptional activity is 60% higher for promoter vectors carrying the rs10459592 C allele compared to those with the A allele (p=0.007), and this polymorphism is associated with HCC risk in non-viral hepatitis cases . CYP19-4 antibody can help elucidate the molecular mechanisms underlying these associations.

What considerations are important when designing experiments to study CYP19 expression in different tissues?

When designing experiments to study CYP19 expression across different tissues:

• Promoter usage: Consider that CYP19 expression is driven by tissue-specific promoters. For liver tumors, expression is primarily regulated by the promoter upstream of CYP19 exon I.6 . Design experiments to capture this tissue-specific regulation.

• Expression level variation: Account for the wide range of expression levels across tissues. Some tissues may require more sensitive detection methods. In amphioxus studies, researchers observed differential expression of cyp19-like genes between male and female gonads, necessitating appropriate controls and quantification methods .

• Sample preparation optimization:

  • For RNA analysis: Use RNase-free conditions and appropriate preservation methods

  • For protein analysis: Optimize extraction buffers for membrane-associated proteins like aromatase

• Multiple detection methods: Employ complementary approaches like real-time PCR, Western blotting, and immunohistochemistry to validate findings.

• Appropriate controls: Include tissue-specific positive and negative controls for accurate interpretation.

• Species differences: Consider evolutionary conservation when working with model organisms. Amphioxus studies have identified two distinct cyp19-like genes with potentially different functions .

• Statistical analysis: Design experiments with sufficient sample sizes to account for biological variation, similar to case-control studies that adjusted for confounding factors like age, sex, race/ethnicity, and lifestyle factors .

How can CYP19-4 antibody contribute to understanding the evolutionary aspects of aromatase function?

CYP19-4 antibody can significantly enhance studies on the evolutionary aspects of aromatase function through:

• Comparative expression analysis: Detecting aromatase protein across different species to map evolutionary conservation and divergence. Recent studies have identified two cyp19-like genes in amphioxus (a primitive chordate), providing insights into the evolutionary origins of vertebrate sex determination mechanisms .

• Structure-function relationships: Identifying conserved epitopes that may represent functionally crucial domains of the protein.

• Developmental expression patterns: Tracking aromatase expression during embryonic development across species to understand its evolving roles.

• Tissue distribution comparison: Mapping expression patterns in homologous tissues across evolutionary distant species to identify core and derived functions.

• Physiological response conservation: Analyzing how aromatase expression responds to similar physiological stimuli across the evolutionary spectrum.

Research on amphioxus has revealed that cyp19-like genes show sex-specific expression patterns in gonads, suggesting conserved roles in reproductive function that predate vertebrate evolution . CYP19-4 antibody that recognizes conserved epitopes can help track these patterns across the evolutionary landscape, potentially identifying when specific functions emerged or diverged.

What are common pitfalls when using CYP19-4 antibody, and how can they be addressed?

Common pitfalls when using CYP19-4 antibody include:

• Non-specific binding: This can be addressed by:

  • Optimizing antibody concentration through titration experiments

  • Using appropriate blocking reagents (5-10% normal serum)

  • Including competing peptides as specificity controls

  • Validating with positive and negative tissue controls

• Variable expression levels: For tissues with low expression:

  • Use signal amplification methods like TSA (tyramide signal amplification)

  • Increase protein loading for Western blots

  • Optimize antigen retrieval methods for immunohistochemistry

• Tissue-specific fixation issues:

  • Different tissues may require modified fixation protocols

  • Formalin-fixed tissues often need antigen retrieval optimization

  • Fresh frozen samples may provide better antigen preservation

• Cross-reactivity with related proteins:

  • Validate antibody specificity against recombinant proteins

  • Consider testing in knockout/knockdown models when available

  • Use multiple antibodies targeting different epitopes for confirmation

• Interference from experimental conditions:

  • Hormone treatments may alter epitope accessibility

  • Certain detergents may affect membrane protein conformation

  • Sample storage conditions can impact antigen integrity

Researchers studying amphioxus cyp19-like proteins developed specific antibodies using carefully selected peptide sequences to ensure specificity, which serves as a model approach for antibody development and validation .

How can CYP19-4 antibody be used effectively in multiplex immunofluorescence studies?

For effective multiplex immunofluorescence with CYP19-4 antibody:

• Primary antibody compatibility:

  • Select primary antibodies from different host species to avoid cross-reactivity

  • If using multiple antibodies from the same species, employ sequential staining with blocking steps

  • Test each antibody individually before multiplexing

• Fluorophore selection:

  • Choose fluorophores with minimal spectral overlap

  • Consider signal intensity matching (brighter fluorophores for less abundant targets)

  • Account for tissue autofluorescence when selecting emission wavelengths

• Optimization protocol:

  • Start with 1:100-1:200 dilution of CYP19-4 antibody

  • Perform antigen retrieval appropriate for all targets

  • Use Tyramide Signal Amplification (TSA) for low-abundance targets

  • Include single-stained controls and unstained controls

• Image acquisition considerations:

  • Capture single-channel images separately to minimize bleed-through

  • Use sequential scanning on confocal microscopes

  • Employ spectral unmixing for closely overlapping fluorophores

• Analysis approaches:

  • Use cell segmentation software for co-expression quantification

  • Apply appropriate thresholding to distinguish specific from non-specific signals

  • Consider proximity analysis for potential protein-protein interactions

These approaches are particularly valuable when studying the relationship between aromatase and other proteins in disease contexts like hepatocellular carcinoma or when examining expression patterns in evolutionary studies .

How does CYP19 genotype affect protein expression and enzymatic activity?

CYP19 genotype significantly impacts protein expression and enzymatic activity through multiple mechanisms:

• Promoter polymorphisms: The rs10459592 A/C polymorphism in the exon I.6 promoter of the CYP19 gene has been shown to affect transcriptional activity. In reporter gene assays, promoter vectors carrying the C allele demonstrated 60% higher transcriptional activity compared to those carrying the A allele (p=0.007) . This directly impacts the level of CYP19 protein produced.

• Coding region variations: Nonsynonymous polymorphisms in the coding region, such as the substitution of arginine for tryptophan at codon 39, can result in non-functional aromatase protein . These structural changes can be detected using specific antibodies that recognize particular epitopes.

• Expression regulation: Different tissues utilize distinct promoters to drive CYP19 expression. In liver tumors, aromatase expression is primarily driven by a promoter upstream of CYP19 exon I.6 . Understanding this tissue-specific regulation is crucial when studying aromatase in different disease contexts.

• Functional consequences: Genotype variations directly translate into differences in estrogen synthesis capacity, with potential implications for hormone-dependent processes and pathologies.

CYP19-4 antibody can be used to assess how these genetic variations manifest at the protein level, providing a direct link between genotype and phenotype in research and clinical studies.

What is the current evidence for CYP19 involvement in hepatocellular carcinoma development?

Current evidence indicates several important roles for CYP19 in hepatocellular carcinoma development:

• Expression alterations: Hepatic aromatase levels and activity are markedly elevated in HCC compared to normal liver tissue . This suggests potential involvement in disease pathogenesis.

• Genetic associations: A dose-dependent association has been observed between the number of high-activity C alleles of the rs10459592 polymorphism in the CYP19 I.6 promoter and risk of non-viral hepatitis-related HCC (p for trend=0.014) . This provides genetic evidence linking aromatase to HCC development.

• Risk stratification: Among subjects negative for at-risk serologic markers of hepatitis B or C, the risk of HCC was significantly higher (odds ratio = 2.25, 95% confidence interval = 1.18–4.31) in individuals homozygous for the C allele compared to those homozygous for the A allele .

• Mechanistic basis: The role of hepatic aromatization of androgen into estrogen provides a potential mechanistic link between aromatase activity and HCC development .

• Population differences: The association between CYP19 polymorphisms and HCC risk has been documented in both high-risk (southern Guangxi, China) and low-risk (US non-Asians) populations, suggesting a fundamental biological relationship rather than a population-specific effect .

These findings support the potential utility of CYP19-4 antibody in diagnostic, prognostic, and therapeutic research related to HCC.

What methodological approaches are recommended for studying the relationship between CYP19 polymorphisms and disease risk?

For robust investigation of CYP19 polymorphisms and disease risk relationships:

• Study design considerations:

  • Case-control studies with carefully matched controls

  • Prospective cohort studies for temporal associations

  • Family-based designs to account for genetic background

  • Cross-population studies to validate findings, as demonstrated in research comparing high-risk Chinese and low-risk US non-Asian populations

• Genotyping approaches:

  • Direct sequencing of PCR products, as employed in studies of CYP19 I.6 promoter polymorphisms

  • High-throughput genotyping methods for larger sample sizes

  • Next-generation sequencing for comprehensive variant detection

• Statistical analysis methods:

  • Adjust for confounding factors (age, gender, race/ethnicity, education level, smoking, alcohol consumption)

  • Test for Hardy-Weinberg equilibrium to validate genotyping quality

  • Perform tests of trend for dose-dependent relationships with allele number

  • Calculate odds ratios using unconditional logistic regression models

  • Conduct interaction analyses between genotype and environmental factors

• Functional validation:

  • Reporter gene assays to assess promoter activity differences

  • Protein expression studies using antibodies like CYP19-4

  • Enzyme activity measurements to link genotype to biochemical phenotype

This multi-faceted approach has successfully revealed associations between CYP19 polymorphisms and HCC risk, as demonstrated in complementary case-control studies that identified a dose-dependent relationship between the C allele of rs10459592 and non-viral hepatitis-related HCC risk .

What are emerging applications of CYP19-4 antibody in precision medicine and biomarker development?

Emerging applications of CYP19-4 antibody in precision medicine and biomarker development include:

• Predictive biomarker development: CYP19-4 antibody can help stratify patients based on aromatase expression levels for targeted therapies. Given the association between CYP19 polymorphisms and HCC risk , antibody-based detection methods could identify patients who might benefit from anti-estrogen therapies.

• Companion diagnostics: Development of standardized immunohistochemical assays using CYP19-4 antibody to guide treatment decisions for aromatase inhibitors in various cancers.

• Liquid biopsy applications: Detection of circulating tumor cells expressing aromatase as a potential minimally invasive biomarker.

• Theranostic approaches: Conjugation of CYP19-4 antibody with imaging agents or therapeutic compounds for targeted delivery to aromatase-expressing tissues.

• Monitoring treatment response: Using quantitative aromatase expression analysis to track the efficacy of hormone-modulating therapies.

• Multi-marker panels: Integration of aromatase detection with other biomarkers for improved diagnostic accuracy, particularly in complex diseases like HCC where multiple factors contribute to pathogenesis .

The identification of CYP19 polymorphisms associated with HCC risk provides a foundation for developing personalized risk assessment tools that could incorporate both genetic testing and antibody-based protein expression analysis .

How might advances in antibody engineering improve CYP19-4 antibody performance in research applications?

Advances in antibody engineering offer several opportunities to enhance CYP19-4 antibody performance:

• Enhanced specificity:

  • Single-domain antibodies (nanobodies) for improved access to conformational epitopes

  • Phage display selection for higher affinity and specificity

  • Site-directed mutagenesis to optimize binding characteristics

• Improved detection sensitivity:

  • Bifunctional antibodies with integrated signal amplification domains

  • Proximity ligation adaptations for super-resolution detection

  • Conjugation to quantum dots or other bright, stable fluorophores

• Versatility enhancements:

  • Recombinant antibody fragments for better tissue penetration

  • Multi-specific antibodies to simultaneously detect CYP19 and interacting proteins

  • Modular design allowing flexible labeling options

• Application-specific modifications:

  • Membrane-permeable antibodies for live-cell imaging

  • pH-sensitive fluorescent conjugates for tracking in different cellular compartments

  • Reversible binding variants for sequential staining protocols

• Reproducibility improvements:

  • Recombinant monoclonal antibody production for batch consistency

  • Standardized validation protocols across multiple applications

  • Epitope mapping to ensure recognition of conserved regions

These advancements would be particularly valuable for evolutionary studies comparing aromatase across species and for detecting subtle expression differences in disease contexts like early HCC development .

What interdisciplinary approaches could benefit from CYP19-4 antibody application?

Innovative interdisciplinary approaches that could benefit from CYP19-4 antibody application include:

• Systems biology integration:

  • Combining antibody-based protein detection with transcriptomics and metabolomics

  • Network analysis of aromatase interactions in health and disease

  • Mathematical modeling of estrogen synthesis pathways informed by quantitative protein data

• Evolutionary developmental biology:

  • Comparative analysis of aromatase expression across species, building on findings in amphioxus and other model organisms

  • Tracking developmental expression patterns to understand conserved functions

  • Correlating genetic variations with protein expression across evolutionary time

• Environmental health sciences:

  • Monitoring aromatase expression changes in response to endocrine-disrupting chemicals

  • Biomonitoring studies in populations with different exposure profiles

  • Assessment of environmental factors that might interact with genetic polymorphisms

• Clinical translation:

  • Integration of genetic testing for CYP19 polymorphisms with antibody-based tissue analysis

  • Development of risk prediction models incorporating both genetic and protein expression data

  • Longitudinal studies correlating aromatase expression with disease progression

• Drug development pipelines:

  • High-content screening using CYP19-4 antibody to identify compounds affecting aromatase expression

  • Target engagement studies for aromatase inhibitor development

  • Tissue-specific drug delivery systems targeting aromatase-expressing cells

These interdisciplinary approaches could significantly advance our understanding of aromatase biology and its implications in diseases like hepatocellular carcinoma, where both genetic factors and protein expression play crucial roles .