CYP4B1 Antibody

What is CYP4B1 and what is its primary biological function?

CYP4B1 belongs to the mammalian cytochrome P450 family 4, subfamily B, polypeptide 1. It functions primarily as an enzyme responsible for the oxidative metabolism of a wide range of endogenous compounds and xenobiotics . This enzyme plays a crucial role in biotransformation processes, particularly in pulmonary tissues where it is predominantly expressed in humans. CYP4B1's enzymatic activity enables the conversion of lipophilic substrates into more water-soluble metabolites, facilitating their elimination from the body. In particular, CYP4B1 has demonstrated high capacity for bioactivating compounds such as 4-Ipomeanol (IPO), a prototypical pulmonary toxin that requires P450-mediated metabolic activation to reactive intermediates to exert its toxic effects .

What is the tissue distribution pattern of CYP4B1 expression?

CYP4B1 exhibits a tissue-specific expression pattern with predominant expression in the lungs of humans . Research using knockout mouse models and gene expression analysis has revealed that CYP4B1 is also expressed in other tissues including the kidney . In experimental models, CYP4B1 expression has been visualized in non-ciliated cells in untreated lung tissue, specifically in bronchioles . Expression levels vary between tissues and can be influenced by various factors including age, exposure to xenobiotics, and disease status. Real-time PCR analysis in wild-type male and female C57Bl/6 adult mice has been used to quantify relative expression levels across different tissues, with lung tissue typically showing the highest expression levels .

What structural information is important when selecting a CYP4B1 antibody?

When selecting a CYP4B1 antibody, researchers should consider several structural factors:

• Binding epitope: Antibodies targeting different regions (N-Terminal, Internal Region, C-Terminal) may yield different results depending on protein folding, post-translational modifications, or protein-protein interactions in the target tissue .

• Sequence specificity: The immunogen sequence is critical - for example, one validated antibody uses a synthetic peptide directed towards the N-terminal region of human CYP4B1 with the sequence "SWAHQFPYAH PLWFGQFIGF LNIYEPDYAK AVYSRGDPKA PDVYDFFLQW" .

• Species cross-reactivity: Different antibodies show varying levels of cross-reactivity with CYP4B1 from different species. Some antibodies demonstrate high predicted reactivity (90-100%) across multiple species including human, mouse, rat, cow, dog, and other mammals .

• Clonality: Most available CYP4B1 antibodies are rabbit polyclonal antibodies, though monoclonal options from mouse hosts are also available for specific applications .

What experimental applications are CYP4B1 antibodies validated for?

CYP4B1 antibodies have been validated for multiple experimental applications:

• Western Blotting (WB): The most common application, with antibodies validated using cell lysates as positive controls . Western blotting can quantify relative protein expression levels across different experimental conditions or tissue types.

• Immunohistochemistry (IHC): Used to visualize the spatial distribution of CYP4B1 in tissue sections, which is particularly valuable for evaluating expression patterns in normal versus diseased tissues .

• Immunofluorescence (IF): Enables co-localization studies with other markers. For example, staining of mouse bronchioles has been performed using CYP4B1 antibody (visualized in red) alongside DAPI (blue) and an acetylated alpha-tubulin specific antibody (cilia, green) .

• Immunoprecipitation (IP): Allows isolation of CYP4B1 protein complexes to study protein-protein interactions .

• ELISA: Provides quantitative measurement of CYP4B1 levels in biological samples .

What are the optimal protocols for immunofluorescence staining with CYP4B1 antibodies?

For optimal immunofluorescence staining with CYP4B1 antibodies, researchers should follow these validated protocols:

• Tissue fixation: Use 4% paraformaldehyde in PBS (pH 7.0). For lung tissues specifically, fix by severing the trachea and injecting fixative down through the airway to expand the lungs, which improves visualization of airways and ensures penetration of staining solutions into terminal bronchioles .

• Antibody dilution: Determine optimal working dilutions experimentally, as recommended by manufacturers .

• Visualization system: Successful staining has been achieved using secondary antibodies conjugated with fluorophores suitable for red channel visualization, paired with DAPI for nuclear counterstaining .

• Critical controls: Include secondary antibody-only negative controls to rule out non-specific staining, as demonstrated in validation experiments where no CYP4B1 staining was observed in control samples .

• Special considerations: When studying pulmonary tissues, co-staining with ciliary markers (such as acetylated alpha-tubulin) can help identify CYP4B1 expression in non-ciliated cells in bronchioles .

How can researchers verify CYP4B1 antibody specificity?

Verifying antibody specificity is crucial for obtaining reliable results. Researchers should implement the following validation approaches:

• Genetic knockout controls: The gold standard for specificity testing is comparing staining between wild-type and CYP4B1 knockout tissues. Pulmonary and renal microsomes prepared from Cyp4b1−/− mice should show no detectable expression of the protein when probed with a specific antibody .

• Peptide competition assays: Pre-incubating the antibody with the immunizing peptide should abolish specific signals in Western blot or immunostaining applications.

• Multiple antibody approach: Using different antibodies targeting distinct epitopes of CYP4B1 and comparing their staining patterns can confirm specificity .

• Correlation with mRNA expression: Comparing protein detection with quantitative RT-PCR data for CYP4B1 expression can provide additional validation .

• Western blot analysis: A specific antibody should detect a single band of the expected molecular weight (approximately 511 AA for human CYP4B1) .

What is the significance of CYP4B1 expression in lung adenocarcinoma?

CYP4B1 has emerged as a significant biomarker in lung adenocarcinoma (LUAD) research:

What molecular mechanisms contribute to altered CYP4B1 expression in cancer?

Several molecular mechanisms have been identified that may contribute to the decreased CYP4B1 expression observed in lung adenocarcinoma:

• Copy number alterations (CNAs): Genomic analysis suggests that CNAs may be one mechanism underlying reduced CYP4B1 expression in tumor tissues .

• DNA methylation: Hypermethylation at specific sites, particularly cg23440155 and cg23414387, has been associated with decreased CYP4B1 expression in cancer samples .

• Gene mutations: The presence of mutations in oncogenes such as KRAS, EGFR, and ALK correlates with altered CYP4B1 expression patterns (p = 0.0239) .

• Disruption of regulatory pathways: Gene set enrichment analysis (GSEA) suggests that CYP4B1 might function in preventing biological metabolism pathways of exogenous and endogenous compounds while enhancing DNA replication and cell cycle activities in cancer cells .

How are CYP4B1 knockout models generated and validated?

Generation and validation of CYP4B1 knockout models involve several critical steps:

• Targeting strategy: Cyp4b1 null mice have been generated following targeted disruption of the gene downstream of exon 1. Embryonic stem cells containing the disrupted Cyp4b1 gene can be obtained from resources such as the knockout mouse project (KOMP) repository .

• Chimeric mouse generation: Embryonic stem cells (15-20) are injected into host blastocysts and implanted into pseudopregnant female mice. Resultant offspring with high coat color chimerism are backcrossed to test for germline transmission .

• Genotyping: Primers are designed to detect both the inserted reporter gene (e.g., lacZ) and the disruption of the Cyp4b1 gene. DNA is isolated from tail snips and processed for PCR amplification with specific cycling conditions (e.g., 95°C for 2 min, 94°C for 30s, 57°C for 30s, 72°C for 2 min, for 35 cycles) .

• Protein expression validation: Western blot analysis comparing wild-type and knockout tissue samples probed for CYP4B1 and other P450 enzymes confirms elimination of CYP4B1 expression while verifying no compensatory upregulation of other P450 isoforms .

• Functional validation: Enzymatic activity assays, such as measuring the bioactivation of 4-Ipomeanol (IPO), confirm functional consequences of CYP4B1 deletion. Pulmonary and renal microsomes from Cyp4b1−/− mice typically show <10% of the bioactivation rates observed in wild-type microsomes .

What approaches can be used to study CYP4B1 tissue distribution patterns?

Researchers can employ several complementary techniques to characterize CYP4B1 tissue distribution:

• Reporter gene expression: In knockout models where lacZ reporter is inserted into the Cyp4b1 locus, tissues can be stained with X-gal to visualize the spatial expression pattern. This requires careful tissue preparation, including appropriate fixation (4% paraformaldehyde/PBS pH 7.0) and staining solution (containing potassium ferricyanide, potassium ferrocyanide, and X-gal) .

• Quantitative RT-PCR: Gene expression across tissues can be determined using the ΔΔCt method with appropriate housekeeping genes like 18S. RNA isolation, cDNA synthesis with random hexamer primers, and amplification using conditions such as 95°C for 10 min followed by 40 cycles of 95°C for 15s, 60°C for 60s allows for relative quantification of expression levels .

• Immunohistochemistry/immunofluorescence: Using validated CYP4B1 antibodies to stain tissue sections provides visual confirmation of protein expression and localization. For lung tissues specifically, CYP4B1 has been detected in non-ciliated cells in untreated bronchioles .

• Western blot analysis: Preparing tissue microsomes by differential centrifugation and analyzing protein expression by Western blot provides quantitative comparison of expression levels across tissues .

How should contradictory CYP4B1 expression data be reconciled across different experimental methods?

When faced with contradictory CYP4B1 expression data, researchers should consider:

• Antibody variability: Different antibodies targeting distinct epitopes may yield varying results depending on protein conformation, post-translational modifications, or epitope accessibility .

• Method-specific limitations: Each detection method has inherent limitations:

  • Western blotting measures total protein but may miss spatial information

  • Immunohistochemistry provides localization but can be less quantitative

  • RT-PCR measures mRNA but may not reflect protein levels due to post-transcriptional regulation

• Sample preparation differences: Variations in tissue fixation, processing, or microsomal preparation can significantly impact results .

• Control validation: Ensure proper positive and negative controls are included in each experiment. For definitive validation, use of knockout tissue controls provides the most reliable reference point .

• Integrated approach: Combining multiple methodologies (protein and mRNA analysis) and using different antibodies targeting distinct epitopes can provide a more complete and reliable picture of CYP4B1 expression.

What factors should be considered when designing experiments to study CYP4B1's role in xenobiotic metabolism?

When investigating CYP4B1's role in xenobiotic metabolism, researchers should consider:

• Model system selection: CYP4B1 function can differ between species, so choosing an appropriate model is critical. Consider using human cell lines or humanized mouse models for translational relevance.

• Substrate selection: CYP4B1 has demonstrated high capacity for bioactivating specific compounds like 4-Ipomeanol (IPO) . Select substrates with known or suspected interaction with CYP4B1.

• Genetic manipulation approaches: Compare results between wild-type systems and those where CYP4B1 is knocked out, knocked down, or overexpressed to establish causality .

• Enzyme kinetics: Analyze both the rate and products of metabolism to fully characterize CYP4B1's role in bioactivation or detoxification pathways.

• Potential compensatory mechanisms: In knockout models, assess whether other P450 isoforms show compensatory upregulation, though studies suggest no evidence of such upregulation in Cyp4b1−/− mice .

• Tissue-specific effects: Given CYP4B1's predominant expression in lungs, special attention should be paid to pulmonary metabolism and toxicity when studying xenobiotic interactions .

How can researchers accurately quantify CYP4B1 expression levels in clinical samples?

For accurate quantification of CYP4B1 in clinical samples, researchers should implement:

• Standardized sample collection and processing: Develop consistent protocols for tissue collection, preservation, and processing to minimize technical variability.

• Multiple quantification methods: Combine protein-based (Western blot, IHC) and mRNA-based (RT-PCR) approaches for comprehensive expression analysis .

• Appropriate reference controls: Include both positive controls (tissues known to express CYP4B1) and negative controls (knockout tissues or tissues with minimal expression) for accurate normalization .

• Digital pathology approaches: For IHC analysis, implement digital image analysis with consistent thresholding to obtain objective quantification of staining intensity and distribution.

• Validation cohorts: Confirm findings across independent patient cohorts to ensure reproducibility of expression patterns and clinical correlations .

• Multi-parameter analysis: Consider CYP4B1 expression in context with other molecular markers to develop comprehensive prognostic or predictive signatures, particularly in cancer research .