CYP84A4 Antibody

What is CYP3A4 and why is it important in research?

CYP3A4 is a major member of the cytochrome P450 enzyme family responsible for the metabolism of approximately 50% of clinically used drugs. This enzyme plays a critical role in pharmaceutical research, toxicology, and clinical pharmacology for several reasons:

• It mediates phase I metabolism of numerous xenobiotics and endogenous compounds

• It contributes significantly to first-pass metabolism in the intestine and liver

• Its activity shows considerable inter-individual variability affecting drug efficacy and safety

• It is involved in many clinically significant drug-drug interactions

• Its expression and activity can be modulated by various compounds through transcriptional regulation

Understanding CYP3A4 function and regulation is essential for predicting drug metabolism, developing new therapeutic agents, and investigating mechanisms of drug toxicity .

What types of CYP3A4 antibodies are available for research applications?

Research-grade CYP3A4 antibodies are available in several formats with distinct properties suitable for different experimental applications:

Polyclonal antibodies like the rabbit anti-CYP3A4 from Chemicon (AB1254) provide broad epitope recognition, while monoclonal antibodies offer specific targeting of defined epitopes, such as the inhibitory MAb 347 that recognizes a region between amino acids 283-504 .

How do I validate CYP3A4 antibody specificity for my experimental system?

Rigorous validation of CYP3A4 antibody specificity is essential for reliable experimental results. Implement these methodological approaches:

First, perform Western blotting with positive controls including human liver microsomes and recombinant CYP3A4 protein to confirm the antibody detects a band of the expected molecular weight (~57 kDa). Include negative controls such as non-expressing tissues or cell lines.

Second, test for cross-reactivity with other CYP3A family members, particularly CYP3A5, which shares high sequence homology with CYP3A4. The literature shows that some antibodies recognize both isoforms while others show preferential binding .

Third, conduct immunodepletion experiments where the antibody is pre-incubated with purified CYP3A4 protein before use in immunodetection to confirm signal specificity.

Fourth, verify antibody performance in your specific application (WB, IHC, etc.) using appropriate controls. Published references in search result demonstrate successful application in Western blotting, immunohistochemistry, and immunofluorescence across various experimental systems.

Finally, consider epitope location when interpreting results, as antibodies recognizing different regions may yield varying results depending on protein conformation or post-translational modifications .

What are the optimal experimental conditions for using CYP3A4 antibodies in Western blotting?

For optimal Western blotting with CYP3A4 antibodies, consider these methodological parameters:

Sample Preparation:

• For microsomal preparations, use standard differential centrifugation methods

• Load 10-20 μg of microsomal protein per lane

• Include appropriate positive controls (human liver microsomes) and negative controls

Electrophoresis and Transfer:

• Use 10-12% SDS-PAGE gels for optimal resolution around 57 kDa (CYP3A4 molecular weight)

• Transfer to PVDF membranes (preferable to nitrocellulose for hydrophobic proteins like CYP3A4)

• Verify transfer efficiency with reversible protein stain before immunodetection

Antibody Incubation:

• Block membranes with 5% non-fat dry milk or BSA in TBST

• For polyclonal antibodies like AB1254, typical dilutions range from 1:1000 to 1:5000

• Incubate primary antibody overnight at 4°C for optimal signal-to-noise ratio

• Use species-appropriate HRP-conjugated secondary antibodies

Detection and Controls:

• Employ ECL detection systems with exposure times optimized for signal intensity

• Include molecular weight markers to confirm target band size

• Run recombinant CYP3A4 as positive control and non-expressing samples as negative controls

• Consider stripping and reprobing for microsomal markers (e.g., calnexin) as loading controls

These conditions have been validated in multiple publications referenced in search result , including studies in various cell lines like C3A, HepaRG, and HepG2 cells.

How can CYP3A4 antibodies be used for epitope mapping studies?

Epitope mapping with CYP3A4 antibodies requires a systematic approach to identify specific binding regions, as demonstrated in published research:

First, generate a series of truncated CYP3A4 constructs expressing different segments of the protein. This can be accomplished by cloning CYP3A4 cDNA fragments into GST expression vectors to create fusion proteins covering various regions of the full-length enzyme .

Second, express these fusion proteins in a suitable system (e.g., bacterial or insect cells) and purify using affinity chromatography. Verify expression by SDS-PAGE and Coomassie staining.

Third, perform Western blotting with the panel of truncated proteins using the antibody of interest. Reactivity with specific fragments indicates the approximate location of the epitope. This approach successfully identified that MAb 347 recognizes a region between amino acids 283-504 on both CYP3A4 and CYP3A5 proteins .

Fourth, refine the epitope mapping by creating smaller overlapping fragments within the initially identified region. This progressive narrowing can pinpoint the epitope to a smaller sequence.

Finally, validate the identified epitope by demonstrating that peptides containing the epitope can compete with the full-length protein for antibody binding. As shown in search result , the construct harboring the epitope can reverse the inhibition of enzymatic activity caused by inhibitory antibodies like MAb 347 .

This methodical approach helps understand the structural basis for antibody recognition and can explain functional effects such as enzyme inhibition.

What controls should be included when using CYP3A4 antibodies in immunohistochemistry?

Robust immunohistochemistry (IHC) with CYP3A4 antibodies requires comprehensive controls to ensure reliable and interpretable results:

Positive Tissue Controls:

• Human liver sections (centrilobular hepatocytes show highest CYP3A4 expression)

• Normal intestinal epithelium (duodenum and jejunum)

• These tissues demonstrate the expected pattern and intensity of CYP3A4 staining

Negative Tissue Controls:

• Tissues known not to express CYP3A4 (e.g., skeletal muscle)

• CYP3A4-negative regions within positive tissues serve as internal controls

Antibody Controls:

• Primary antibody omission (to assess secondary antibody specificity)

• Isotype control or pre-immune serum at equivalent concentration

• Antibody pre-absorption with recombinant CYP3A4 protein (to confirm specificity)

Technical Controls:

• Serial dilutions of primary antibody to determine optimal concentration

• Different antigen retrieval methods to optimize epitope accessibility

• Positive control slides processed in parallel with each experimental batch

Validation Controls:

• Correlation with other detection methods (e.g., mRNA expression by qPCR or in situ hybridization)

• Comparison of staining patterns using antibodies targeting different epitopes

• For novel applications (e.g., detecting CYP3A4 in brain tissue), additional validation is essential

Research cited in search result successfully employed CYP3A4 antibodies for immunohistochemistry in various tissues, including studies examining CYP3A4 expression in epileptic brain tissue, demonstrating the versatility of properly validated antibodies .

How do inhibitory CYP3A4 antibodies work and how can they be used in research?

Inhibitory CYP3A4 antibodies provide powerful tools for studying enzyme function through specific binding that impairs catalytic activity. Their mechanisms and applications include:

Inhibitory Mechanisms:

• Binding to substrate recognition sites, preventing substrate access

• Inducing conformational changes that alter the active site geometry

• Interfering with electron transfer from redox partners

• Blocking protein-protein interactions essential for activity

Experimental Applications:

• Reaction phenotyping: Determine CYP3A4 contribution to metabolism of specific substrates by selective inhibition. MAb 347 inhibited quinine 3-hydroxylation in human liver microsomes by >70%, allowing quantification of CYP3A4's contribution to this pathway .

• Isoform discrimination: Some antibodies show differential inhibition of CYP3A family members, like MAb 347 which potently inhibits CYP3A5 (95% at ≤0.20 mg IgG/nmol P450) but only moderately inhibits CYP3A4 at higher concentrations .

• Structure-function studies: Correlating epitope locations with inhibitory potency provides insights into functional domains. The identification of amino acids 283-504 as the binding region for inhibitory MAb 347 highlights the importance of this region for catalytic function .

• In vitro drug interaction studies: Inhibitory antibodies can model protein-specific inhibition without the confounding effects of chemical inhibitors, which may affect multiple enzymes.

When using these antibodies, researchers should establish inhibition curves by titrating antibody concentrations against enzyme activity, include appropriate controls (non-specific IgG), and consider the potential differential effects on various CYP3A4 substrates .

How can CYP3A4 antibodies be adapted for high-content imaging assays?

High-content imaging with CYP3A4 antibodies enables quantitative analysis of protein expression, localization, and correlation with cellular phenotypes. Implementing this approach requires:

Cell Model Selection:

• HepaRG cells provide a physiologically relevant model with stable CYP3A4 expression

• Cryopreserved differentiated HepaRG cells offer consistency between experiments

• HepG2 and C3A cells may require induction of CYP3A4 expression

Assay Development:

• Optimization of immunostaining protocols:

  • Fixation method (4% paraformaldehyde typically preserves epitope accessibility)

  • Permeabilization conditions (0.1-0.2% Triton X-100 for intracellular access)

  • Antibody concentration (titrate to maximize signal-to-noise ratio)

  • Incubation times and temperatures

• Multiplexing strategies:

  • Combine CYP3A4 staining with organelle markers (e.g., ER, mitochondria)

  • Include cell health indicators (e.g., mitochondrial membrane potential with JC-1 dye)

  • Add nuclear counterstains for cell identification and normalization

Image Acquisition and Analysis:

• Acquire images at appropriate magnification (20-40x) for single-cell resolution

• Develop segmentation algorithms to identify individual cells and subcellular compartments

• Extract multiple parameters (intensity, texture, localization) for comprehensive analysis

• Normalize CYP3A4 signals to cell number or area for quantitative comparisons

Validation and Applications:

• Correlate immunofluorescence signal with functional CYP3A4 activity

• Use the assay to screen compounds for CYP3A4 induction or inhibition

• Apply to hepatotoxicity studies, as referenced in search result regarding automated detection of hepatotoxic compounds

This approach has been successfully implemented in studies examining CYP3A4 expression and localization in response to various treatments, as documented in publications cited in search result .

How do CYP3A4 antibodies perform in different subcellular fractionation preparations?

The performance of CYP3A4 antibodies varies across different subcellular fractionation preparations, requiring specific methodological considerations:

Microsomal Fractions:

• Optimal preparation for CYP3A4 detection as the enzyme is predominantly localized to the endoplasmic reticulum

• Western blotting with polyclonal antibodies typically yields strong signals at the expected 57 kDa

• Use microsomal markers (e.g., calnexin) as loading controls rather than cytosolic proteins

• Recommended protein loading: 5-20 μg per lane for high-expressing tissues like liver

Mitochondrial Fractions:

• Some CYP3A4 may be detected in mitochondrial preparations due to ER contamination or actual mitochondrial localization

• Higher protein loading (20-50 μg) may be necessary for detection

• Essential to verify fraction purity with markers for mitochondria (e.g., VDAC) and ER (e.g., calnexin)

Cytosolic Fractions:

• Typically negative for CYP3A4 (useful as a negative control)

• Any detection in cytosolic fractions should be interpreted with caution and verified for potential microsomal contamination

Whole Cell Lysates:

• Less sensitive than enriched microsomal fractions for CYP3A4 detection

• Higher background may necessitate more stringent washing conditions

• Useful for relative comparisons but less optimal for absolute quantification

Membrane Raft Preparations:

• Detergent-resistant membrane preparations can contain CYP3A4

• Require specialized extraction protocols and higher antibody concentrations

For optimal results across these preparations, polyclonal antibodies like the one described in search result often provide superior sensitivity due to recognition of multiple epitopes, while monoclonal antibodies offer greater specificity but may be more sensitive to epitope accessibility issues in different preparation methods .

How can CYP3A4 antibodies be used to study the enzyme's role in drug-induced liver injury?

CYP3A4 antibodies provide valuable tools for investigating the enzyme's role in drug-induced liver injury (DILI) through several methodological approaches:

Expression Analysis in DILI Models:

• Immunohistochemistry in liver tissues:

  • Examine zonal distribution of CYP3A4 in relation to injury patterns

  • Compare expression in injured versus adjacent normal tissue

  • Correlate with markers of cellular stress and inflammation

• Western blotting in experimental systems:

  • Monitor CYP3A4 protein levels following drug exposure

  • Assess time-dependent changes in expression during injury development

  • Compare parent drug effects versus metabolite-induced changes

Mechanistic Investigations:

• Metabolic inhibition studies:

  • Use inhibitory antibodies like those described in search result to block CYP3A4 activity

  • Determine if CYP3A4 inhibition prevents or exacerbates drug toxicity

  • This approach helps distinguish between parent drug toxicity and metabolite-mediated injury

• Protein adduct detection:

  • Immunoprecipitate CYP3A4 using specific antibodies

  • Analyze for covalent modifications by reactive metabolites

  • Identify potential mechanisms of mechanism-based inhibition or enzyme inactivation

High-Content Imaging Applications:

• Multiplexed analysis in hepatocyte models:

  • Combine CYP3A4 immunostaining with viability markers

  • Track changes in subcellular localization during toxic response

  • Correlate CYP3A4 expression with mitochondrial dysfunction

  • This approach has been validated in HepaRG cells as referenced in search result

• Quantitative assessment of hepatotoxicity:

  • Develop image-based algorithms correlating CYP3A4 expression patterns with toxicity

  • Perform dose-response and time-course analyses

  • Compare responses across different hepatocyte models with varying CYP3A4 expression

These approaches enable researchers to determine whether CYP3A4 plays a protective role (through detoxification) or contributes to injury (through bioactivation) for specific compounds, providing critical insights for drug development and safety assessment .

What factors affect CYP3A4 antibody epitope recognition and how can these be addressed?

Multiple factors can influence CYP3A4 antibody epitope recognition, potentially affecting experimental outcomes. Understanding and addressing these factors is crucial for reliable results:

Protein Conformation:

• Native versus denatured states expose different epitopes

• For applications requiring detection of native CYP3A4 (e.g., immunoprecipitation), select antibodies validated under non-denaturing conditions

• For Western blotting, most antibodies perform well with denatured protein, as seen with the polyclonal antibody in search result

Post-Translational Modifications:

• Phosphorylation, glycosylation, or ubiquitination may mask epitopes

• If modifications are suspected to interfere with detection, treat samples with appropriate enzymes (phosphatases, glycosidases) before analysis

• Use multiple antibodies targeting different regions to provide complementary information

Fixation Effects in Microscopy:

• Crosslinking fixatives (formaldehyde) may mask epitopes

• Perform antigen retrieval (heat or enzymatic) to restore epitope accessibility

• Optimize fixation time to balance structural preservation with epitope accessibility

Detergent Solubilization:

• As a membrane protein, CYP3A4 requires detergent for solubilization

• Different detergents may affect protein conformation and epitope exposure

• Test multiple detergents (Triton X-100, CHAPS, sodium deoxycholate) to optimize extraction

Species Differences:

• The polyclonal antibody in search result shows reactivity to human and porcine CYP3A4

• When working with animal models, verify cross-reactivity with the species-specific CYP3A enzyme

• Consider species-specific amino acid differences in the epitope region

Methodological Solutions:

• Use epitope-mapped antibodies with known binding regions, like MAb 347 described in search result

• Employ multiple antibodies targeting different epitopes for confirmation

• Include positive controls processed identically to experimental samples

• Optimize sample preparation for each specific application

• For critical applications, validate findings with complementary non-antibody-based methods

How can CYP3A4 antibodies be used to study protein-protein interactions in drug metabolism pathways?

CYP3A4 antibodies provide powerful tools for investigating protein-protein interactions crucial to drug metabolism pathways. These methodological approaches offer complementary insights:

Co-Immunoprecipitation (Co-IP):

• Use CYP3A4 antibodies to pull down the enzyme and associated proteins from liver microsomes

• Identify interaction partners by Western blotting or mass spectrometry

• Compare interactions under different conditions (e.g., substrate presence, inhibitor treatment)

• This approach can detect associations with electron transfer partners (cytochrome P450 reductase, cytochrome b5) and other regulatory proteins

Proximity Ligation Assay (PLA):

• Combine CYP3A4 antibodies with antibodies against potential interaction partners

• The PLA technique generates fluorescent signals only when proteins are in close proximity (<40 nm)

• This enables visualization of interactions in situ within cells or tissue sections

• Quantify interaction frequency and localization under various experimental conditions

Immunofluorescence Co-Localization:

• The polyclonal antibody referenced in search result has been validated for immunofluorescence

• Perform dual immunostaining with CYP3A4 and potential interaction partners

• Use confocal microscopy to assess spatial overlap at subcellular resolution

• Calculate co-localization coefficients for quantitative analysis

Functional Interaction Studies:

• Utilize inhibitory antibodies like those described in search result

• Determine if antibody binding affects interactions with other proteins

• Compare the effects of antibodies targeting different epitopes

• This approach helped establish that the region between amino acids 283-504 is functionally important in CYP3A4/5

Cross-Linking Mass Spectrometry:

• Use chemical cross-linkers to stabilize transient protein-protein interactions

• Immunoprecipitate CYP3A4 complexes using specific antibodies

• Identify cross-linked peptides by mass spectrometry

• Map interaction interfaces at amino acid resolution

These techniques have revealed important interactions between CYP3A4 and various proteins involved in drug metabolism, transport, and cellular regulation, contributing to our understanding of the complex network governing xenobiotic metabolism .

What approaches can be used to develop new inhibitory antibodies against CYP3A4?

Developing effective inhibitory antibodies against CYP3A4 requires strategic approaches to generate reagents that specifically block enzymatic activity:

Immunization Strategies:

• Antigen selection:

  • Use properly folded, full-length recombinant CYP3A4 to preserve conformational epitopes

  • Consider peptide immunogens from regions known to be involved in substrate binding or catalysis

  • The region between amino acids 283-504 has proven effective for generating inhibitory antibodies

• Host selection:

  • Rabbits produce high-affinity polyclonal antibodies (like the one in search result )

  • Mice or rats are suitable for monoclonal antibody development

  • Consider species differences in CYP3A enzymes when selecting immunization hosts

Screening Methodologies:

• Primary screening:

  • Test antibody binding to CYP3A4 by ELISA or Western blotting

  • Confirm specificity against related CYP3A family members

• Functional screening:

  • Develop metabolism assays using CYP3A4-specific substrates like quinine

  • Screen for inhibition of enzymatic activity in reconstituted systems or microsomes

  • Establish dose-dependent inhibition curves for promising candidates

  • MAbs 347, 351, 352, 354, and 357 were identified as inhibitory using this approach

Characterization and Refinement:

• Epitope mapping:

  • Use truncation constructs to identify binding regions as demonstrated in search result

  • Fine-map epitopes using peptide arrays or alanine scanning mutagenesis

  • Correlate epitope location with inhibitory potency

• Specificity optimization:

  • Test cross-reactivity with other CYP enzymes

  • Evaluate species specificity (e.g., human vs. animal CYP3A enzymes)

  • Characterize differential inhibition patterns (e.g., MAb 347 shows different potency against CYP3A4 vs. CYP3A5)

• Antibody engineering:

  • Generate Fab or scFv fragments to improve tissue penetration

  • Consider bispecific formats combining CYP3A4 targeting with another functionality

These approaches have successfully yielded inhibitory antibodies with research applications in reaction phenotyping, mechanistic studies, and differential inhibition of CYP3A family members .

How do CYP3A4 antibodies perform in detecting the protein in non-hepatic tissues?

Detecting CYP3A4 in non-hepatic tissues presents unique challenges and requires specific methodological considerations:

Brain Tissue:

• Search result references studies successfully detecting CYP3A4 in human epileptic brain tissue

• Cellular localization studies revealed expression at the blood-brain barrier

• Immunohistochemistry and immunofluorescence techniques were effective for visualization

• Studies demonstrated functional significance of CYP3A4 in the epileptic brain

Intestinal Tissue:

• CYP3A4 is expressed at high levels in enterocytes, particularly in the duodenum and jejunum

• Antibodies perform well in detecting the protein in intestinal epithelium

• Cryosections often provide better epitope preservation than formalin-fixed paraffin-embedded samples

• Consider gradient of expression along the intestinal tract when interpreting results

Methodological Adaptations:

• Sample preparation:

  • Optimize fixation protocols for each tissue type

  • For tissues with lower expression, increase sample loading in Western blotting

  • Consider using more sensitive detection systems (e.g., amplification steps in IHC)

• Validation approaches:

  • Always include positive controls (liver tissue sections or lysates)

  • Confirm specificity with RNA expression data (RT-PCR or in situ hybridization)

  • Use multiple antibodies targeting different epitopes for confirmation

• Detection sensitivity:

  • Polyclonal antibodies like the one in search result often provide better sensitivity for tissues with lower expression

  • Signal amplification techniques may be necessary for tissues with very low expression

  • Consider concentration steps in sample preparation (e.g., microsomal isolation)

• Analysis considerations:

  • Quantify relative expression compared to liver (standard reference tissue)

  • Account for tissue-specific background and autofluorescence

  • Correlate protein detection with functional activity when possible

These adaptations have enabled researchers to successfully detect and characterize CYP3A4 in various non-hepatic tissues, revealing important roles beyond traditional drug metabolism in the liver .

What are the considerations for using CYP3A4 antibodies in studying enzyme regulation?

CYP3A4 antibodies are valuable tools for investigating regulatory mechanisms controlling this enzyme's expression and activity. Several methodological considerations apply:

Transcriptional Regulation Studies:

• Protein-DNA interaction analysis:

  • Combine chromatin immunoprecipitation with CYP3A4 antibodies to study transcription factor binding

  • Correlate protein expression changes with transcriptional activity

  • Search result references studies examining upregulation of CYP3A4 expression through novel transcriptional regulators

• Nuclear receptor interactions:

  • Investigate pregnane X receptor (PXR) and constitutive androstane receptor (CAR) effects on CYP3A4

  • Studies cited in search result examined PXR-independent, CAR-related mechanisms of CYP3A4 induction

  • Western blotting with CYP3A4 antibodies provides quantitative assessment of induction responses

Post-Translational Regulation:

• Protein stability and turnover:

  • Use pulse-chase experiments combined with immunoprecipitation

  • Assess effects of various treatments on CYP3A4 protein half-life

  • Investigate ubiquitination and proteasomal degradation pathways

• Enzyme inhibition mechanisms:

  • Studies with inhibitory antibodies like MAb 347 provide insights into functional regulation

  • Compare substrate-dependent effects with antibody inhibition patterns

  • This approach has revealed important structural determinants of enzymatic activity

Experimental Systems:

• Cell model selection:

  • HepaRG cells provide a physiologically relevant model with stable CYP3A4 expression

  • HepG2 and C3A cells may require induction of CYP3A4 expression

  • Multiple studies referenced in search result utilized these models successfully

• Induction protocols:

  • Treat cells with known CYP3A4 inducers (rifampicin, phenobarbital)

  • Monitor protein expression changes by Western blotting

  • Correlate with enzymatic activity for functional validation

• Transduction approaches:

  • Study cited in search result used "transfection with a novel chimeric regulator" to upregulate CYP3A4

  • Assess effects on protein expression using specific antibodies

  • This approach reveals mechanisms of transcriptional regulation

These methodologically rigorous approaches enable comprehensive investigation of the complex regulatory networks controlling CYP3A4 expression and activity, with important implications for drug metabolism, drug interactions, and personalized medicine .