CYP19-1 Antibody

What is CYP19A1 and why is it important in research?

CYP19A1 encodes the enzyme aromatase, which plays a crucial role in the biosynthesis of estrogens from androgens by catalyzing the conversion of testosterone to estradiol in various tissues. This enzyme represents a critical component in endocrine and paracrine signaling pathways that regulate reproductive development, bone health, and neurological function. Research interest in CYP19A1 has expanded significantly due to its implications in hormonal cancers, metabolic disorders, and more recently, its association with severe COVID-19 outcomes specifically in male patients. The enzyme has been found to be highly expressed in the lungs of deceased male COVID-19 patients, with a prevalence of 81.3% in autopsy studies . Understanding aromatase biology through targeted antibody applications allows researchers to investigate tissue-specific expression patterns and functional variations that may contribute to pathophysiological processes.

What tissue types show significant CYP19A1 expression detectable by antibodies?

CYP19A1 demonstrates a distinctive tissue distribution pattern that varies significantly between sexes and across developmental stages. Immunohistochemical studies using validated CYP19A1 antibodies have successfully detected expression in human placenta, brain, kidney, skeletal muscle, testis, spleen, lung, and ovarian tissues . In COVID-19 research specifically, CYP19A1 was found to be abundantly expressed in epithelial and endothelial cells, but most profoundly in macrophages in the lungs of deceased COVID-19 male patients . This expression pattern was consistent across three independent study sites (Hamburg, Tübingen, and Rotterdam). When designing immunostaining experiments, researchers should consider tissue-specific fixation requirements and potential cross-reactivity with other P450 enzymes. Positive control tissues, particularly human placenta which shows robust CYP19A1 expression, should be included in experimental designs to validate antibody performance and specificity in the researcher's specific experimental conditions.

What are the recommended dilutions for CYP19A1 antibody across different applications?

Optimal antibody dilution varies significantly depending on the specific application, tissue type, and detection method employed. For Western blot applications, a dilution range of 1:500-1:1000 has been validated for the 16554-1-AP CYP19A1 antibody when working with cell lysates from A2780 cells or human placental tissue extracts . For immunohistochemistry applications, a broader dilution range of 1:50-1:500 is recommended, with the important caveat that antigen retrieval methods significantly impact signal quality. Researchers should note that TE buffer at pH 9.0 is the preferred antigen retrieval method, though citrate buffer at pH 6.0 has been reported as an acceptable alternative . For immunofluorescence applications, both paraffin sections (IF-P) and cell cultures (IF/ICC) typically require 1:50-1:500 dilutions. It is critically important to emphasize that these recommendations serve as starting points, and antibody performance may vary significantly between laboratories based on sample preparation methods, detection systems, and specific research questions being addressed.

How do I troubleshoot weak or absent signal when using CYP19A1 antibody in Western blotting?

Troubleshooting weak or absent CYP19A1 signals requires systematic evaluation of several technical parameters. First, ensure adequate protein loading (50-100 μg of total protein is often necessary for detecting endogenous aromatase) and verify transfer efficiency through Ponceau S staining of membranes. Consider that CYP19A1 expression varies dramatically between tissues, with highest expression in placenta, ovary, and testis; therefore, positive control lysates from these tissues are recommended for initial validation. If signal remains weak, optimize extraction buffers to effectively solubilize membrane-associated proteins, as CYP19A1 is predominantly localized to the endoplasmic reticulum . Membrane blocking conditions warrant particular attention, with 5% non-fat milk often yielding superior results compared to BSA-based blockers. Extended primary antibody incubation (overnight at 4°C) frequently improves detection sensitivity. Additionally, enhanced chemiluminescence (ECL) reagents with longer exposure times may be necessary to visualize bands. For tissues with expected low expression, consider enrichment strategies such as microsomal fraction isolation to concentrate the target protein before Western blot analysis.

How can I verify CYP19A1 antibody specificity in my experimental system?

Verifying antibody specificity is fundamental to generating reliable research data and requires implementation of multiple complementary approaches. Researchers should begin by comparing staining/detection patterns with published expression profiles across tissues and cell types. A key validation approach involves parallel analysis of samples with known differential expression, such as comparing placental tissue (high expression) with muscle tissue (lower expression). For definitive validation, genetic approaches including siRNA knockdown or CRISPR-Cas9 mediated knockout of CYP19A1 provide robust controls to confirm antibody specificity. These genetic approaches should produce observable reduction or elimination of signal in Western blot, immunocytochemistry, or other detection methods. Peptide competition assays, where pre-incubation of the antibody with excess immunizing peptide blocks specific binding, offer additional verification. Researchers investigating CYP19A1 variants should be particularly vigilant, as mutations in exons, such as the R435C and F234del variants, may alter antibody epitope recognition . Cross-validation using multiple antibodies targeting different epitopes provides additional confidence in specificity when resources permit.

What are the key considerations for subcellular localization studies of CYP19A1?

Subcellular localization studies of CYP19A1 require careful attention to both technical parameters and biological factors that influence aromatase distribution. CYP19A1 is predominantly localized to the endoplasmic reticulum, which can be confirmed through co-localization studies with ER markers such as calnexin . For immunofluorescence approaches, optimization of cell fixation methods is critical; paraformaldehyde (4%) fixation for 15-20 minutes typically preserves CYP19A1 antigenicity while maintaining cellular architecture. Permeabilization conditions significantly impact detection sensitivity, with 0.1-0.2% Triton X-100 generally providing adequate access to the ER-associated enzyme. When designing GFP fusion constructs for live-cell imaging, researchers should consider that N-terminal tagging may interfere with ER targeting, while C-terminal tagging might disrupt catalytic function. For co-localization studies, confocal microscopy with appropriate controls for bleed-through is strongly recommended over widefield fluorescence. Researchers investigating CYP19A1 variants should note that certain mutations may affect protein localization; for example, studies using GFP-tagged constructs demonstrated that wild-type aromatase and the R435C mutant localized to the endoplasmic reticulum, while the exon5del mutant showed a more diffuse cytoplasmic and nuclear distribution pattern .

How can I investigate functional consequences of CYP19A1 genetic polymorphisms?

Investigating the functional impact of CYP19A1 polymorphisms requires a multi-faceted approach combining genetic, biochemical, and cellular techniques. Site-directed mutagenesis of human aromatase cDNA in expression vectors allows recreation of clinically identified variants, such as the R435C point mutation or exon deletions that have been documented in research literature . Transient transfection of these constructs into appropriate cell lines (typically HEK293 or COS7 cells) enables comparative analysis of enzyme function. Activity assays measuring the conversion of radiolabeled substrates (e.g., [1β-³H]androstenedione) to estrogen provide quantitative assessment of catalytic efficiency. Enzyme kinetics should be determined by generating Lineweaver-Burk plots (1/v versus 1/[S]) to calculate Km and Vmax parameters, allowing distinction between altered substrate affinity and maximal catalytic capacity . For variants affecting protein stability rather than catalytic function, pulse-chase experiments with cycloheximide treatment can reveal differences in protein half-life. The use of GFP-tagged constructs facilitates visualization of potential alterations in subcellular localization that might contribute to functional deficits. Additionally, researchers should consider that certain polymorphisms, such as rs6493497 and rs7176005 in the 5'-flanking region, may influence transcriptional regulation rather than protein function directly, necessitating reporter gene assays to fully characterize their impact .

What methodological approaches should be used when studying the relationship between CYP19A1 expression and disease outcomes?

Methodological rigor in studying CYP19A1-disease associations requires careful consideration of experimental design, statistical approaches, and potential confounding variables. Researchers should implement case-control designs with appropriately matched subjects, particularly concerning age, sex, and hormonal status, as these factors significantly influence aromatase expression. Quantitative assessment of CYP19A1 expression should employ multiple complementary techniques, including qRT-PCR for mRNA quantification, Western blotting for protein levels, and immunohistochemistry for tissue localization patterns. When investigating genetic associations, researchers should conduct comprehensive genotyping rather than focusing on isolated SNPs, as demonstrated in studies examining CYP19A1 polymorphisms in breast cancer patients receiving aromatase inhibitor therapy . Statistical analyses must account for linkage disequilibrium between polymorphisms, with tools such as Haploview software for calculating D' and R² values, and haplo.stats for haplotype estimation and comparison among phenotypes . For disease outcome studies, as exemplified in COVID-19 research, researchers should employ multiple independent cohorts (such as the Hamburg, Tübingen, and Rotterdam autopsy cohorts) to validate findings, while implementing blinded assessment protocols to minimize investigator bias . Additionally, when examining hormone-dependent outcomes, sensitive assay methods such as gas chromatographic negative ionization tandem mass spectrometry (GC/NCI/MS/MS) and liquid chromatographic electrospray tandem mass spectrometry (LC/ESI/MS/MS) should be used for accurate quantification of estrone and estradiol levels .

How can I optimize immunohistochemical detection of CYP19A1 in formalin-fixed, paraffin-embedded tissues?

Optimizing immunohistochemical detection of CYP19A1 in FFPE tissues requires careful attention to multiple technical parameters that significantly impact staining specificity and sensitivity. Antigen retrieval represents the most critical step; while the manufacturer recommends TE buffer at pH 9.0, optimization experiments comparing multiple retrieval methods (including citrate buffer pH 6.0) should be conducted for each specific tissue type . Retrieval duration and temperature significantly impact epitope accessibility, with 20-30 minutes at 95-98°C typically providing optimal results for CYP19A1. Primary antibody concentration should be titrated for each tissue type and lot of antibody, beginning with the recommended 1:50-1:500 dilution range . Overnight incubation at 4°C often improves signal-to-noise ratio compared to shorter incubations at room temperature. Signal amplification systems warrant careful selection; polymer-based detection methods generally offer superior sensitivity compared to avidin-biotin systems while reducing background. For tissues with high lipofuscin content (particularly brain tissues), additional steps to quench autofluorescence are essential. When analyzing staining patterns, researchers should reference the established CYP19A1 expression profile, expecting predominant signal in epithelial and endothelial cells, with particularly strong expression in macrophages as observed in lung tissues . Validation through multiple controls is essential: positive controls should include placental tissue, while negative controls should include both antibody omission and tissues known to lack aromatase expression.

What are the key considerations when investigating CYP19A1's role in COVID-19 pathophysiology?

Investigating CYP19A1's role in COVID-19 requires careful attention to sex-specific differences and methodological consistency across multiple research platforms. Male-female differences represent a critical focus area, as research has demonstrated that CYP19A1 is abundantly expressed in the lungs of men who died of COVID-19 with a prevalence of 81.3%, while detectable expression in females was limited to cases where SARS-CoV-2 antigen or RNA remained present . When designing immunohistochemistry studies, researchers should implement cell-type specific markers to identify CYP19A1 expression in epithelial cells, endothelial cells, and particularly macrophages, where expression appears most pronounced. Viral detection should be performed in consecutive sections to correlate CYP19A1 expression with viral persistence. Quantitative real-time PCR approaches should target specific CYP19A1 transcripts, as studies have shown dramatic upregulation (up to 40-fold) specifically in SARS-CoV-2 infected cells compared to other respiratory viruses . When investigating genetic associations, particular attention should be paid to the CYP19A1 Thr201Met variant, which has been identified as having 68.7% penetrance in severely ill male COVID-19 patients . Translational research should consider both direct viral effects on CYP19A1 expression and the potential therapeutic implications of aromatase inhibition, as preclinical evidence suggests CYP19A1 inhibition may improve long-term lung health in SARS-CoV-2-infected male animal models .

How should researchers approach CYP19A1 antibody-based studies in hormone-responsive cancers?

Hormone-responsive cancer research using CYP19A1 antibodies demands rigorous methodology to generate clinically relevant insights. Researchers must first establish the relationship between CYP19A1 expression and tumor characteristics through carefully designed tissue microarray studies incorporating diverse tumor grades, stages, and molecular subtypes. Antibody validation in this context should include comparison with enzymatic activity assays measuring aromatase function directly. When investigating potential predictive or prognostic value of CYP19A1 expression, researchers should implement scoring systems that account for both staining intensity and percentage of positive cells, with independent assessment by multiple pathologists to ensure reproducibility. The subcellular localization pattern provides important information, as alterations from the typical endoplasmic reticulum pattern may indicate dysfunction. Sample collection timing requires careful consideration, particularly in neoadjuvant studies where pre- and post-treatment samples allow assessment of treatment effects on aromatase expression . For translational relevance, CYP19A1 antibody studies should be integrated with genomic data, including analysis of polymorphisms such as rs6493497 and rs7176005 in the 5'-flanking region of CYP19A1 exon 1.1, which have been associated with aromatase activity changes following inhibitor treatment . Additionally, researchers should consider implementing digital pathology approaches for quantitative assessment of CYP19A1 staining to overcome limitations of subjective interpretation in conventional scoring methods.

What methodological approaches are recommended for investigating CYP19A1 antibody cross-reactivity with variant proteins?

Investigating CYP19A1 antibody cross-reactivity with variant proteins requires systematic characterization of epitope recognition across genetic variants. Researchers should begin by identifying the specific epitope recognized by the antibody; for example, some commercial antibodies target the C-terminal region (codons 376-390) , which would be unaffected by variants in other regions but potentially compromised by C-terminal mutations. Expression vectors containing wild-type and variant CYP19A1 sequences (including point mutations, deletions, and splice variants) should be transfected into appropriate cell lines for controlled comparison of antibody recognition patterns. Western blot analysis can reveal not only presence/absence of signal but also potential alterations in apparent molecular weight, as demonstrated in studies of the exon5del variant which results in a protein approximately 6 kDa smaller than wild-type aromatase . Immunoprecipitation followed by mass spectrometry provides definitive identification of the protein being recognized. For commercial antibodies lacking detailed epitope information, peptide arrays containing overlapping sequences spanning the entire aromatase protein can identify specific binding regions. Researchers investigating specific variants, such as the R435C mutation or F234del, should be particularly vigilant about potential epitope alterations . Additionally, computational approaches predicting protein structural changes resulting from genetic variants can provide insights into potential epitope disruption, guiding experimental design focused on specific protein regions of interest.

How should researchers design experiments to investigate tissue-specific differences in CYP19A1 expression?

Experimental design for investigating tissue-specific CYP19A1 expression patterns requires careful consideration of sampling strategy, tissue processing methods, and detection techniques to generate meaningful comparative data. Researchers should implement comprehensive sampling approaches that capture both traditional high-expression tissues (placenta, gonads) and those with more context-dependent expression (brain, adipose, bone, lung) to establish a complete expression profile. Tissue acquisition timing is critically important, particularly for reproductive tissues where aromatase expression fluctuates with hormonal cycles; therefore, precise documentation of menstrual/estrous cycle stage, pregnancy status, or age is essential. Preservation methods significantly impact detection sensitivity; for optimal results, researchers should process matched tissue samples using parallel fixation protocols (formalin, frozen sections, and fresh tissue for protein/RNA extraction) to enable multimodal analysis. Quantitative assessment should employ both mRNA-based methods (qRT-PCR with validated primers spanning exon-exon junctions) and protein-based approaches (Western blotting and immunohistochemistry) to distinguish between transcriptional and post-transcriptional regulation. Cell-type specific expression patterns, as observed in lung tissue where macrophages show particularly high CYP19A1 expression , necessitate co-localization studies with lineage-specific markers. For comprehensive analysis, researchers should consider implementing laser capture microdissection to isolate specific cell populations for subsequent molecular analysis, enabling precise correlation between cellular identity and aromatase expression levels.

What controls should be included when validating novel CYP19A1 antibodies for research applications?

Rigorous validation of novel CYP19A1 antibodies requires implementation of a comprehensive control framework addressing specificity, sensitivity, and reproducibility across multiple experimental platforms. Positive tissue controls should include human placenta, which consistently demonstrates high aromatase expression, while negative controls should include tissues with minimal documented expression, such as specific muscle groups . Genetic validation represents the gold standard approach, requiring comparison of staining patterns between wild-type samples and those with CYP19A1 knockdown/knockout. Researchers developing novel antibodies should perform side-by-side comparisons with established commercial antibodies across multiple applications (Western blot, IHC, IF) to demonstrate concordance or explain discrepancies. Epitope mapping is essential for mechanistic interpretation, particularly when studying genetic variants; this can be accomplished through peptide arrays or truncation constructs to identify the specific recognition sequence. Cross-reactivity assessment must include related P450 enzymes with structural similarity to aromatase. For monoclonal antibodies, isotype-matched control antibodies against irrelevant antigens should be tested in parallel to identify potential non-specific binding. Inter-laboratory reproducibility should be established through blinded sample exchange and standardized protocols. Additionally, antibody performance should be validated across species if cross-reactivity is claimed; the 16554-1-AP antibody, for example, has demonstrated reactivity with human, mouse, and rat samples , but species-specific optimization may be necessary.

How can researchers effectively combine genetic and protein expression studies to comprehensively characterize CYP19A1 function?

Integrative approaches combining genetic and protein-level analyses provide the most comprehensive characterization of CYP19A1 function across physiological and pathological contexts. Researchers should begin with genetic sequencing of both coding regions and regulatory elements, as polymorphisms in both domains have been associated with functional consequences . When characterizing novel variants, site-directed mutagenesis to recreate mutations in expression constructs allows direct assessment of functional impact through activity assays and protein characterization studies. Protein expression analyses should employ both quantitative (Western blot) and spatial (immunohistochemistry, immunofluorescence) approaches to determine both abundance and localization patterns. Enzyme kinetic studies measuring the conversion of androgen substrates to estrogens provide crucial information on catalytic efficiency, allowing researchers to distinguish between alterations in substrate affinity (Km) and maximal reaction velocity (Vmax) . For variants affecting regulatory regions, reporter gene assays incorporating wild-type and variant promoter sequences can identify alterations in transcriptional regulation, as demonstrated in studies of rs6493497 and rs7176005 polymorphisms . Chromatin immunoprecipitation (ChIP) assays further characterize transcription factor binding affected by regulatory variants. For a systems-level understanding, researchers should correlate CYP19A1 genotype with hormone levels (estrone, estradiol) using sensitive mass spectrometry-based methods , while considering the influence of other enzymes in steroidogenic pathways. This multilevel approach allows distinction between genetic variations affecting transcription, translation, protein stability, subcellular localization, or catalytic function.