CYP2A6V2 Antibody

What is CYP2A6V2 and what biological function does it detect?

CYP2A6V2 polyclonal antibody detects endogenous levels of CYP2A6V2 protein, which is a member of the cytochrome P450 superfamily of enzymes. The CYP2A6 gene encodes monooxygenases that catalyze numerous reactions involved in drug metabolism and the synthesis of cholesterol, steroids, and other lipids. This protein localizes to the endoplasmic reticulum and its expression is induced by phenobarbital. Functionally, the enzyme hydroxylates coumarin and metabolizes various compounds including nicotine, aflatoxin B1, nitrosamines, and several pharmaceuticals .

The antibody recognizes the human CYP2A6 protein, which was formerly referred to as CYP2A3 before being renamed. It's part of a large cluster of cytochrome P450 genes from the CYP2A, CYP2B, and CYP2F subfamilies located on chromosome 19q .

What is the molecular weight of the CYP2A6V2 protein detected by this antibody?

The CYP2A6V2 protein detected by this antibody has an observed molecular weight of approximately 56 kDa when analyzed by Western blot techniques. This molecular weight is consistent across validated antibody products from different manufacturers . Researchers should note this observed band size when validating their experimental results and confirming specific detection of the target protein.

What species reactivity has been confirmed for CYP2A6V2 antibody?

The CYP2A6V2 polyclonal antibody exhibits confirmed reactivity with human samples, which is the primary species for which this antibody has been developed and validated. Some commercial antibodies also demonstrate cross-reactivity with rat and mouse samples, making them versatile tools for comparative studies across these mammalian models . When considering research involving other species, preliminary validation experiments should be conducted to confirm reactivity.

What are the validated applications for CYP2A6V2 antibody?

The CYP2A6V2 polyclonal antibody has been validated for multiple experimental applications including:

• Western Blotting (WB): For protein detection and quantification

• Immunohistochemistry on paraffin-embedded tissues (IHC-p): For localizing protein in tissue sections

• Immunofluorescence (IF): For cellular localization studies

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection

Each application requires specific optimization of antibody dilution and experimental conditions. For Western blot applications, a dilution range of 1:500 to 1:2000 is typically recommended. For immunohistochemistry applications, dilutions between 1:100 and 1:300 are generally effective. ELISA applications may require higher dilutions up to 1:20000 for optimal results .

How should researchers optimize CYP2A6V2 antibody dilutions for different applications?

Optimization of antibody dilution is critical for successful experiments with CYP2A6V2 polyclonal antibody. A methodological approach includes:

For immunohistochemistry, antigen retrieval using Tris-EDTA buffer at pH 9.0 has shown effective results. Secondary antibody dilution around 1:200 at room temperature for 30 minutes is generally appropriate . Researchers should always include proper positive and negative controls to validate specificity.

What are the recommended storage conditions for maintaining CYP2A6V2 antibody activity?

CYP2A6V2 polyclonal antibody is typically supplied in a liquid formulation containing PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide as preservatives . For optimal stability and activity:

• Store the antibody at -20°C for long-term storage (stability typically guaranteed for 1 year)

• Avoid repeated freeze-thaw cycles as these can diminish antibody activity

• For short-term use (1-2 weeks), storage at 4°C is acceptable

• If necessary, prepare working aliquots to minimize freeze-thaw cycles

The concentrated antibody (typically at 1 mg/ml) should maintain ≥90% purity when stored properly. Always centrifuge the antibody briefly before use to collect the entire volume at the bottom of the tube .

How can researchers verify the specificity of CYP2A6V2 antibody for their experimental system?

Verifying antibody specificity is essential for reliable experimental results. A comprehensive validation approach includes:

• Western blot validation: Look for a single band at the expected molecular weight (56 kDa) in tissues known to express CYP2A6V2 (primarily liver)

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples; this should abolish specific signals

• Positive control selection: Include samples from tissues known to express high levels of CYP2A6 (liver tissues)

• Knockout/knockdown validation: Compare signal in wild-type versus CYP2A6-deficient samples (if available)

• Cross-reactivity assessment: Test against closely related CYP family members to confirm specificity

The antibody is typically generated against a synthesized peptide derived from the internal region of human CYP2A6V2 and purified through affinity chromatography using an epitope-specific immunogen . Understanding the specific epitope region can help predict potential cross-reactivity.

How do genetic polymorphisms in CYP2A6 affect antibody detection and experimental interpretation?

CYP2A6 is known for significant genetic polymorphism across populations. These variations can impact antibody detection and experimental interpretation in several ways:

• Allelic variants and poor metabolizer phenotypes: Individuals with certain allelic variants exhibit "poor metabolizer phenotypes," meaning they do not efficiently metabolize substrates like coumarin or nicotine . These genetic variations may affect protein structure and potentially epitope recognition.

• Population-specific variations: Research has documented unique genetic variations in CYP2A6 across different populations that contribute to inter-individual variations in drug metabolism and response .

• Experimental considerations:

  • When using human samples, genetic background may influence protein detection levels

  • Variant-specific antibodies may be required for certain research questions

  • Researchers should consider genotyping samples when quantitative comparisons are critical

  • Western blot band patterns may vary based on the presence of splice variants or post-translational modifications affected by genetic polymorphisms

For comprehensive studies involving drug metabolism, combining antibody-based protein detection with genetic analysis provides the most complete understanding of CYP2A6 status and function.

What approaches can be used to design antibodies with enhanced specificity for CYP2A6V2?

Recent advances in antibody engineering allow researchers to design antibodies with enhanced specificity profiles. For CYP2A6V2, several approaches have demonstrated promise:

• Phage display selection: This technique can be used to generate antibodies with specific high affinity for CYP2A6V2 while excluding binding to closely related cytochrome P450 family members .

• Biophysics-informed modeling: By using data from phage display experiments, computational models can identify different binding modes associated with specific ligands, enabling the design of antibodies that either:

  • Target only CYP2A6V2 with specific high affinity

  • Exhibit controlled cross-specificity for multiple desired targets

• High-throughput sequencing combined with machine learning: This approach has demonstrated the ability to make predictions beyond experimentally observed sequences, allowing for customized specificity profiles .

• Counter-selection strategies: Computational methods can efficiently eliminate off-target antibodies, addressing a major challenge in antibody development .

These approaches require specialized expertise but offer significant advantages for research requiring precise discrimination between very similar epitopes.

What are common issues encountered with CYP2A6V2 antibody in Western blotting and how can they be resolved?

Western blotting with CYP2A6V2 antibody may present several technical challenges. Here are common issues and their methodological solutions:

For optimal results with CYP2A6V2 detection, researchers should:

• Prepare microsomal fractions for enrichment of endoplasmic reticulum proteins

• Use freshly prepared samples with appropriate protease inhibitors

• Consider extended transfer times for the relatively large 56 kDa protein

• Include positive controls from tissues with known high expression (liver tissue)

What factors influence CYP2A6V2 detection in immunohistochemistry applications?

Several critical factors can influence the success of CYP2A6V2 detection in immunohistochemistry:

• Fixation conditions: Overfixation can mask epitopes; formalin fixation for 24-48 hours is typically optimal

• Antigen retrieval method: Tris-EDTA buffer at pH 9.0 has been validated for effective retrieval of CYP2A6V2 epitopes in paraffin sections

• Tissue-specific considerations:

  • CYP2A6 is predominantly expressed in the liver

  • Expression can be induced by phenobarbital and other compounds

  • Basal expression levels may vary between individuals due to genetic polymorphisms

• Procedural optimizations:

  • Primary antibody incubation at 4°C overnight often yields better results than shorter incubations

  • Secondary antibody dilution around 1:200 at room temperature for 30 minutes is generally appropriate

  • Visualization systems should be selected based on expression level (DAB for moderate-high expression; amplification systems for low expression)

• Specificity controls:

  • Include tissues known to be negative for CYP2A6 expression

  • Consider peptide competition controls

  • Compare with alternative antibodies targeting different epitopes of the same protein

How can researchers differentiate between CYP2A6 and closely related cytochrome P450 family members?

Distinguishing CYP2A6 from other closely related cytochrome P450 family members requires careful experimental design:

• Epitope selection: Understanding the specific epitope recognized by the antibody is crucial. The CYP2A6V2 polyclonal antibody typically targets synthesized peptides derived from the internal region of human CYP2A6V2 , which should be compared with sequences of related family members to predict potential cross-reactivity.

• Validation approaches:

  • Western blotting in tissues with differential expression patterns of CYP family members

  • Recombinant protein controls for each potential cross-reactive family member

  • Immunodepletion studies with specific family members

  • Comparison with mRNA expression data (RT-PCR) for correlation

• Computational prediction tools: Leveraging biophysics-informed models trained on experimentally selected antibodies can help predict and analyze potential cross-reactivity .

• Functional assays: Combining immunodetection with functional assays specific for CYP2A6 activity (e.g., coumarin 7-hydroxylation) can provide additional confirmation of specificity.

How might CYP2A6V2 antibodies contribute to personalized medicine approaches?

CYP2A6V2 antibodies have significant potential to advance personalized medicine through several applications:

• Pharmacogenetic profiling: CYP2A6 plays a crucial role in metabolizing drugs like efavirenz used in HIV treatment. Genetic variations in CYP2A6 contribute to inter-individual variations in patient responses . Antibodies that can detect variant-specific protein expression could help stratify patients beyond genotyping alone.

• Protein-level biomarker development: While genetic testing identifies variants, antibody-based detection can quantify actual protein expression levels, potentially offering more direct correlation with metabolic capacity.

• Therapeutic monitoring applications: Development of point-of-care diagnostic tools using CYP2A6V2 antibodies could help monitor therapeutic efficacy and adjust dosing for drugs metabolized by this enzyme.

• Integration with emerging technologies:

  • Combination with mass spectrometry for absolute quantification of protein variants

  • Implementation in microfluidic/lab-on-chip platforms for rapid clinical testing

  • Integration with machine learning approaches to predict drug responses based on protein expression patterns

As initiatives like the MedeA Initiative expand to incorporate pharmacogenetic variations into prescriptive practices globally, including in African populations where such initiatives are currently limited , antibody-based detection methods will serve as important complementary tools to genetic testing.

What novel methodologies might enhance CYP2A6V2 antibody design and application in the future?

Emerging research points to several promising methodologies that could enhance CYP2A6V2 antibody design and application:

• Binding mode identification: Biophysics-informed models that identify different binding modes associated with particular ligands allow for computational design of antibodies with customized specificity profiles .

• Machine learning integration: High-throughput sequencing combined with machine learning enables predictions beyond experimentally observed sequences, potentially allowing for antibodies with unprecedented specificity .

• Synthetic biology approaches:

  • Designer epitopes that enhance specificity for particular CYP2A6 variants

  • Recombinant expression systems for variant-specific antibody generation

  • CRISPR-edited cell lines for validation of variant-specific detection

• Improved selection methods:

  • Counter-selection strategies that can be implemented computationally rather than experimentally to eliminate off-target binding

  • Phage display experiments with increasingly complex selection parameters

  • High-throughput functional screening platforms

These advances promise to expand the utility of CYP2A6V2 antibodies in both research and clinical applications, particularly in fields requiring discrimination between very similar epitopes or variants.

How can CYP2A6V2 antibodies contribute to understanding population-specific pharmacogenetic variations?

CYP2A6V2 antibodies offer valuable tools for investigating population-specific pharmacogenetic variations:

• Population-stratified protein expression analysis: Beyond genetic polymorphisms, antibody-based detection can reveal actual protein expression levels across different populations, potentially identifying post-transcriptional factors that influence enzyme activity.

• Integration with genomic data: Studies focusing on African populations have identified variations in CYP genes related to treatment responses in patients with HIV . Combining antibody-based protein detection with genomic data provides a more comprehensive understanding of how genetic variations manifest at the protein level.

• Methodological approaches for population studies:

  • Tissue microarrays with population-diverse samples

  • Correlation of protein expression with genetic variants

  • Functional studies correlating protein detection with metabolic activity

• Translational research applications:

  • Development of population-optimized dosing guidelines

  • Identification of high-risk groups for adverse drug reactions

  • Personalized medicine approaches for specific populations

This research direction is particularly important as most pharmacogenetic studies have historically focused on European populations, leaving significant knowledge gaps regarding other population groups .