CYP19-2 Antibody

What is CYP19 and why is it important in research?

CYP19 (aromatase) is a cytochrome P450 enzyme responsible for converting androgens to estrogens. This enzyme plays critical roles in various physiological and pathological processes, including hormone-dependent cancers, reproductive biology, and metabolism. In hepatic tissues, CYP19 has been linked to hepatocellular carcinoma (HCC) development, with evidence suggesting that increased aromatase expression and activity may promote HCC in non-virally infected individuals . The enzyme is nearly undetectable in healthy adult liver tissues but shows elevated expression in certain pathological conditions, making it an important research target.

What experimental applications are optimal for CYP19-2 antibody?

CYP19-2 antibody can be utilized in multiple experimental approaches:

• Western blotting: For quantitative assessment of aromatase protein levels in tissue or cell lysates

• Immunohistochemistry (IHC): For visualizing aromatase distribution in tissue sections (fixed or frozen)

• Immunofluorescence (IF): For subcellular localization studies and co-localization with other proteins

• Immunoprecipitation: For isolating aromatase and studying protein-protein interactions

When selecting an application, researchers should consider the research question and sample type. For example, when studying tissue-specific aromatase expression patterns in HCC samples, IHC would be preferred to visualize distribution between tumor and surrounding tissues .

How do I determine appropriate antibody dilutions for my experiment?

Determining optimal antibody dilution requires systematic titration:

• Begin with manufacturer's recommended dilution range

• Perform a dilution series (e.g., 1:250, 1:500, 1:1000, 1:2000)

• Include appropriate positive controls (tissues or cells known to express CYP19)

• Include negative controls (tissues lacking CYP19 or using secondary antibody only)

• Evaluate signal-to-noise ratio at each dilution

• Select the dilution providing maximum specific signal with minimal background

The optimal dilution may vary based on experimental conditions, sample type, and detection system. Polyclonal CYP19 antibodies may require different dilution protocols than monoclonal variants due to their broader epitope recognition.

What controls should be included when working with CYP19-2 antibody?

Robust experimental design requires appropriate controls:

Positive controls:

• Cell lines with confirmed CYP19 expression (e.g., HepG2, Huh7 for liver research)

• Tissues known to express high levels of aromatase (placenta, ovary, adipose tissue)

Negative controls:

• Cell lines lacking CYP19 expression (some HCC cell lines like HA22T have been reported to be aromatase-deficient)

• Primary antibody omission control

• Isotype control antibody

• Blocking peptide competition (pre-incubation of antibody with immunizing peptide)

Technical controls:

• Loading control for Western blotting (e.g., β-actin, GAPDH)

• Tissue processing controls for IHC (parallel sections with established antibodies)

How should samples be prepared for optimal CYP19-2 antibody detection?

Sample preparation is critical for successful antibody-based detection:

For Western blotting:

• Use fresh or properly frozen tissue/cells

• Include protease inhibitors in lysis buffer

• Optimize protein extraction conditions for membrane proteins

• Consider using specialized detergents (e.g., CHAPS, NP-40)

• Determine appropriate protein loading amount (typically 20-50 μg)

• Optimize denaturation conditions (temperature, reducing agents)

For IHC/IF:

• Fixation: 10% neutral buffered formalin (24-48 hours)

• Proper tissue processing and embedding

• Optimization of antigen retrieval method:

  • Heat-induced epitope retrieval (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Enzymatic retrieval (proteinase K)

• Blocking of endogenous peroxidase activity and non-specific binding sites

The selection of appropriate methods should be validated for each experimental system.

How can CYP19-2 antibody be used to investigate CYP19 polymorphisms associated with disease risk?

CYP19 polymorphisms have been associated with various diseases, including hepatocellular carcinoma. Research approaches include:

• Combined genotype-phenotype analysis:

  • Genotype subjects for CYP19 polymorphisms (e.g., the exon I.6 promoter A/C polymorphism)

  • Use CYP19-2 antibody to quantify protein expression in tissues from individuals with different genotypes

  • Correlate protein levels with genotype and disease status

• Functional analysis of promoter variants:

  • Create reporter constructs containing different CYP19 promoter variants

  • Transfect cells and measure transcriptional activity

  • Use CYP19-2 antibody to confirm protein expression differences

• Tissue-specific expression analysis:

  • Perform IHC using CYP19-2 antibody on tissue microarrays from patients with known genotypes

  • Quantify expression patterns across different tissues and disease stages

  • Correlate with clinical outcomes

In a study examining CYP19 I.6 promoter polymorphism, researchers found that the C allele drove higher aromatase expression and was associated with increased non-viral hepatitis-related HCC risk, suggesting that increased aromatase expression promotes HCC development in non-virally infected individuals .

What techniques can be used to simultaneously analyze CYP19 at protein and genetic levels?

Integrated analysis approaches include:

• Combined immunohistochemistry and in situ hybridization:

  • Perform IHC with CYP19-2 antibody to detect protein

  • Follow with in situ hybridization to detect CYP19 mRNA

  • Compare protein and mRNA localization in the same tissue section

• Chromatin immunoprecipitation (ChIP) analysis:

  • Use antibodies against transcription factors known to regulate CYP19

  • Immunoprecipitate protein-DNA complexes

  • Sequence or PCR-amplify bound DNA fragments to identify binding sites

  • Correlate with CYP19 protein expression using CYP19-2 antibody

• Laser capture microdissection with proteomics and genomics:

  • Isolate specific cell populations from tissue sections

  • Perform proteomic analysis with CYP19-2 antibody for protein detection

  • Parallel genetic analysis for CYP19 variants

  • Integrate data to understand tissue-specific regulation

What are common causes of weak or inconsistent CYP19-2 antibody signal?

Several factors can contribute to suboptimal antibody performance:

How can researchers distinguish between true aromatase signal and non-specific staining?

Differentiating specific from non-specific signals requires multiple validation approaches:

• Parallel analysis with different antibodies:

  • Use multiple CYP19 antibodies targeting different epitopes

  • Compare staining patterns for consistency

• Correlation with known expression patterns:

  • Compare antibody staining with established tissue expression data

  • Verify expected subcellular localization (primarily endoplasmic reticulum for CYP19)

• RNA-protein correlation:

  • Compare protein detection with CYP19 mRNA levels by RT-qPCR or RNA-seq

  • Discrepancies may indicate post-transcriptional regulation or antibody issues

• Genetic models:

  • Use CYP19 knockout/knockdown models as negative controls

  • Use CYP19 overexpression models as positive controls

How should researchers quantify and statistically analyze CYP19 expression patterns?

Quantification and statistical analysis depend on the experimental approach:

For Western blotting:

• Use appropriate software (ImageJ, Image Lab, etc.) for densitometry

• Normalize to loading controls

• Compare relative expression between experimental groups

• Apply appropriate statistical tests (t-test, ANOVA) based on experimental design

For IHC quantification:

• Establish scoring criteria (e.g., H-score, Allred score)

• Consider both staining intensity and percentage of positive cells

• Use digital image analysis software for objective quantification

• Employ appropriate statistical methods, adjusting for confounding variables

In epidemiological studies, logistic regression models can be used to assess associations between CYP19 expression and disease risk, adjusting for factors such as age, sex, ethnicity, and other relevant variables .

How can researchers interpret contradictory results between CYP19 mRNA and protein levels?

Discrepancies between mRNA and protein levels are common and may reflect important biological mechanisms:

• Post-transcriptional regulation:

  • Investigate microRNA regulation of CYP19 mRNA

  • Assess mRNA stability differences between experimental groups

• Post-translational modifications:

  • Use phospho-specific antibodies to assess CYP19 phosphorylation

  • Investigate ubiquitination and other modifications affecting protein stability

• Methodological considerations:

  • Evaluate sample preparation differences between RNA and protein analyses

  • Assess antibody specificity and detection sensitivity

  • Consider temporal dynamics (mRNA changes may precede protein changes)

• Tissue heterogeneity:

  • Use single-cell approaches to resolve cell type-specific expression patterns

  • Employ laser capture microdissection for targeted analysis of specific regions