CYP71B23 Antibody

What is CYP71B23 and what is its role in plant systems?

CYP71B23 is a cytochrome P450 enzyme that belongs to the CYP71 family, one of the largest P450 families in plants. In Arabidopsis thaliana, CYP71B23 has been identified among 22 highly overrepresented cytochrome P450 enzymes in metabolic pathways related to plant defense mechanisms . The enzyme participates in specialized metabolite biosynthesis, particularly in pathways associated with camalexin production, which is a key phytoalexin involved in plant immune responses to pathogen attack. Unlike other well-characterized P450s such as CYP71A13 and CYP71B15 (PAD3), which have confirmed roles in the camalexin biosynthetic pathway, the precise biochemical function of CYP71B23 is still being elucidated.

How does CYP71B23 interact with other cytochrome P450 enzymes in metabolic pathways?

CYP71B23 has been observed to accumulate significantly alongside other P450 enzymes in protein complexes associated with camalexin biosynthesis . Research suggests that it may form part of a metabolon—a transient multi-enzyme complex that facilitates efficient channeling of metabolites through biosynthetic pathways. While direct protein-protein interactions between CYP71B23 and enzymes like CYP71B15 or CYP71A13 have not been as extensively characterized as other interactions (e.g., CYP71A13 with CYP71B15), its co-localization within the endoplasmic reticulum (ER) membrane suggests potential cooperative functions in specialized metabolite biosynthesis.

What technical characteristics define a CYP71B23 antibody?

CYP71B23 antibodies are immunological reagents developed specifically against epitopes of the Arabidopsis thaliana CYP71B23 protein. The commercially available antibody is a polyclonal antibody that recognizes multiple epitopes on the CYP71B23 protein . These antibodies are typically generated by immunizing host animals with either the full-length recombinant protein or specific peptide sequences unique to CYP71B23. The polyclonal nature provides robust recognition across multiple protein conformations, making them valuable tools for detecting native CYP71B23 in plant tissues under various experimental conditions.

What are the optimal methods for using CYP71B23 antibodies in protein localization studies?

For effective protein localization studies, researchers should implement a multi-faceted approach combining biochemical fractionation with microscopy techniques. Begin with subcellular fractionation to separate ER membranes (where most P450 enzymes localize) from other cellular components. Western blotting of these fractions using the CYP71B23 antibody at a 1:1000 dilution can confirm presence in the ER fraction. For immunofluorescence microscopy, fix Arabidopsis tissue samples with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100, and block with 3% BSA before incubating with the primary antibody overnight at 4°C. Co-staining with established ER markers like RFP-HDEL is essential for confirming localization, similar to methods that have successfully demonstrated the ER localization of related enzymes such as CYP71A12, CYP71A13, and CYP71B15 .

How can CYP71B23 antibodies be used to investigate protein-protein interactions in metabolon formation?

To investigate CYP71B23's role in metabolon formation, implement co-immunoprecipitation (co-IP) protocols similar to those used for other P450 enzymes in the camalexin pathway. Challenge Arabidopsis plants with pathogens like Botrytis cinerea to induce expression of defense-related proteins, then harvest tissue 24-48 hours post-infection. Solubilize microsomes with 1% digitonin to preserve protein-protein interactions, and perform IP using anti-CYP71B23 antibodies coupled to protein A/G beads. Analyze precipitated proteins by mass spectrometry to identify interaction partners . For validation of specific interactions, express CYP71B23 with suspected partners (such as CYP71A13 or CYP71B15) as epitope-tagged proteins in Nicotiana benthamiana, followed by targeted co-IP experiments. This approach has successfully revealed interactions between other P450 enzymes involved in camalexin biosynthesis.

What is the recommended protocol for detecting CYP71B23 expression changes during pathogen infection?

To effectively monitor CYP71B23 expression changes during pathogen infection, implement a time-course experiment using both protein and transcript analysis. Inoculate Arabidopsis plants with relevant pathogens such as Botrytis cinerea or Plectosphaerella cucumerina, and collect samples at 0, 12, 24, 48, and 72 hours post-infection. For protein analysis, prepare total protein extracts from infected tissues using a buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100, and protease inhibitors. Perform Western blot analysis using the CYP71B23 antibody, normalized against a constitutive protein like actin. Complement protein data with qRT-PCR analysis of CYP71B23 transcript levels using gene-specific primers. This dual approach provides insights into both transcriptional and post-transcriptional regulation of CYP71B23 during immune responses, similar to expression patterns observed for other defense-related P450 enzymes .

How can functional genomics approaches be combined with CYP71B23 antibody studies to elucidate its precise role in plant metabolism?

Implementing a comprehensive functional genomics strategy requires integrating antibody-based protein studies with genetic and metabolomic analyses. Generate CYP71B23 knockout mutants and CYP71B23-overexpressing lines in Arabidopsis thaliana, then confirm protein absence or overexpression using the CYP71B23 antibody through Western blotting. Challenge these plants with pathogens and perform metabolic profiling using liquid chromatography-mass spectrometry (LC-MS) to identify metabolites that significantly differ between wild-type and modified plants. Focus particularly on indole derivatives and related compounds in the camalexin pathway. Additionally, perform co-expression analysis with known camalexin biosynthetic genes such as CYP71A13 and CYP71B15 under various biotic stresses. Consider creating double and triple mutants with other P450 enzymes, similar to studies with cyp71A12, cyp71A13, and cyp82C2 mutants , to identify potential functional redundancy or synergistic relationships.

What strategies can resolve contradictory results when CYP71B23 antibody detection conflicts with transcriptomic data?

When facing discrepancies between protein detection using CYP71B23 antibodies and transcriptomic data, a systematic troubleshooting approach is required. First, validate antibody specificity by testing against recombinant CYP71B23 protein and protein extracts from confirmed cyp71B23 knockout plants. For post-transcriptional regulation analysis, measure CYP71B23 mRNA stability through actinomycin D chase experiments and assess protein turnover rates using cycloheximide treatments. Examine potential miRNA-mediated regulation by identifying predicted miRNA binding sites in the CYP71B23 transcript and quantifying candidate miRNAs during the experimental conditions. Investigate protein stability through MG132 proteasome inhibitor treatments to determine if discrepancies result from accelerated protein degradation despite robust transcription. Finally, consider technical factors such as antibody affinity, protein extraction efficiency from membrane fractions, and potential epitope masking in protein complexes or due to post-translational modifications.

How can CYP71B23 antibodies be used in comparative analyses across different plant species?

For cross-species comparative analysis using CYP71B23 antibodies, first perform in silico analysis to identify CYP71B23 homologs in target species through phylogenetic analysis of P450 sequences. Test antibody cross-reactivity by performing Western blots on protein extracts from selected plant species, beginning with closely related Brassicaceae family members before expanding to more distant relatives. For species where cross-reactivity is confirmed, compare CYP71B23 homolog expression patterns during pathogen challenge or abiotic stress conditions to identify conserved or divergent responses. Complement antibody studies with heterologous expression of identified homologs in yeast or E. coli systems, followed by in vitro enzyme assays to compare substrate specificities and catalytic efficiencies. This integrative approach helps establish functional conservation or specialization of CYP71B23-like enzymes across plant lineages and provides insights into the evolution of specialized metabolic pathways in different plant families.

What critical controls should be included when using CYP71B23 antibodies in immunoprecipitation experiments?

When designing immunoprecipitation experiments with CYP71B23 antibodies, several critical controls must be included to ensure result validity. First, include a negative control using pre-immune serum or IgG from the same species as the CYP71B23 antibody was raised in to identify non-specific binding. Second, incorporate a cyp71B23 knockout mutant as a biological negative control to verify antibody specificity. For co-immunoprecipitation studies investigating protein-protein interactions, include reciprocal precipitations where available antibodies against suspected interaction partners (such as CYP71A13) are used for pulldown, followed by CYP71B23 detection. Additionally, test the stability of interactions under different detergent conditions (0.1-1% digitonin, NP-40, or Triton X-100) to determine optimal solubilization conditions that preserve protein-protein interactions while effectively extracting membrane-bound proteins. Finally, perform competition assays with recombinant CYP71B23 protein to confirm binding specificity, similar to methods used in studying interactions between other P450 enzymes involved in camalexin biosynthesis .

How should researchers interpret Western blot data when multiple bands appear using CYP71B23 antibodies?

When multiple bands appear in Western blots using CYP71B23 antibodies, systematic analysis is required to determine their identities. First, compare observed band sizes with the predicted molecular weight of CYP71B23 (approximately 58 kDa), accounting for potential post-translational modifications. For bands of higher molecular weight, test whether they represent dimers or oligomers by including reducing agents like DTT or β-mercaptoethanol at increased concentrations. For lower molecular weight bands, evaluate whether they might be degradation products by adding additional protease inhibitors during extraction or by performing extraction at 4°C versus room temperature. To identify potential splice variants, compare protein data with RT-PCR analysis using primers spanning different exon junctions of the CYP71B23 gene. For closely related P450 family members that might cross-react, perform peptide competition assays using the immunogenic peptide (if a peptide antibody was used) to identify specific versus non-specific bands. Additionally, create an epitope-tagged version of CYP71B23 and express it in plants to compare migration patterns with the endogenous protein detected by the antibody.

What experimental design is optimal for studying CYP71B23 involvement in stress responses using both transcriptomic and proteomic approaches?

An optimal experimental design for studying CYP71B23 involvement in stress responses requires parallel transcriptomic and proteomic analyses across multiple time points and stress conditions. Begin with a factorial design exposing Arabidopsis plants to biotic stressors (such as B. cinerea, P. cucumerina) and abiotic stressors (drought, cold, salinity) with samples collected at 0, 6, 12, 24, 48, and 72 hours post-treatment. For transcriptomics, perform RNA-seq or targeted qRT-PCR focusing on CYP71B23 and related metabolic pathway genes. For proteomics, implement both targeted Western blot analysis using CYP71B23 antibodies and untargeted LC-MS/MS proteomics on microsomal fractions to capture membrane-associated protein dynamics. Include phosphoproteomics analysis to identify potential regulatory post-translational modifications of CYP71B23. Create parallel experiments with cyp71B23 knockout plants and complemented lines to establish causal relationships between gene function and observed metabolic changes. This multi-omics approach, similar to studies conducted on related enzymes like CYP71A12 , allows for comprehensive mapping of CYP71B23's role in various stress response networks while accounting for potential post-transcriptional regulatory mechanisms.

How does CYP71B23 functionally compare to other characterized P450 enzymes in the camalexin biosynthetic pathway?

How can researchers use CYP71B23 antibodies to investigate potential metabolon formation during plant immune responses?

Investigating metabolon formation requires a sophisticated experimental approach combining structural and functional analyses. Begin by using CYP71B23 antibodies for in situ proximity ligation assays (PLA) to visualize protein-protein interactions within plant cells during immune responses. This technique can confirm close physical proximity (<40 nm) between CYP71B23 and other pathway components such as CYP71A13 or CYP71B15. Follow with blue native PAGE of solubilized microsomes to preserve protein complexes, then perform Western blotting with the CYP71B23 antibody to identify higher molecular weight complexes that may represent metabolons.

For dynamic studies, implement bimolecular fluorescence complementation (BiFC) by creating split-fluorescent protein fusions with CYP71B23 and potential interaction partners, then observe complex formation during pathogen challenge. Complement these approaches with substrate channeling assays to determine if metabolon formation enhances pathway efficiency. Finally, use chemical crosslinking coupled with immunoprecipitation using CYP71B23 antibodies followed by mass spectrometry to identify all components of the putative metabolon, similar to approaches that have successfully identified interactions between CYP71A13 and CYP71B15 in the camalexin biosynthetic pathway .

What methodological approaches can determine if CYP71B23 is involved in alternative metabolic pathways beyond camalexin biosynthesis?

To explore CYP71B23's potential involvement in alternative metabolic pathways, implement a comprehensive metabolomics approach coupled with genetic manipulation. Generate CYP71B23 overexpression lines and knockout mutants, then perform untargeted metabolomics using high-resolution LC-MS/MS comparing these lines to wild-type plants under both basal and stress-induced conditions. Pay particular attention to indole derivatives, triterpenoids, and other specialized metabolites frequently processed by CYP71 family enzymes.

Use stable isotope labeling with precursors like 13C-tryptophan to track metabolic flux through various pathways in wild-type versus cyp71B23 mutant plants. For substrate identification, express recombinant CYP71B23 in yeast or insect cell systems, then perform in vitro enzyme assays with potential substrates identified from metabolomics data. Validate in planta function through complementation studies of cyp71B23 mutants with the wild-type gene and analyze restoration of metabolic profiles. Additionally, perform co-expression network analysis using publicly available transcriptome data to identify genes consistently co-regulated with CYP71B23 across diverse conditions, which may indicate involvement in shared metabolic pathways beyond the well-studied camalexin biosynthetic route .

What are common challenges in detecting CYP71B23 protein in plant tissues and how can they be overcome?

Detecting CYP71B23 protein in plant tissues presents several challenges that require specific technical adaptations. First, as a membrane-associated P450 enzyme, CYP71B23 may be difficult to extract efficiently using standard protein extraction buffers. Implement microsomal preparation protocols using ultracentrifugation (100,000 × g for 1 hour) after initial homogenization to concentrate membrane proteins. For solubilization, use mild detergents like 0.5-1% digitonin or 1% Triton X-100 to maintain protein integrity while releasing membrane-bound proteins.

Second, expression levels may be low or tissue-specific under basal conditions - induce expression with pathogen treatment (e.g., Botrytis cinerea or flg22 peptide) for 24-48 hours before protein extraction. Third, sample preparation is critical - include reducing agents (5 mM DTT) and a comprehensive protease inhibitor cocktail to prevent degradation. For Western blotting, longer transfer times (16 hours at 30V) may be necessary for efficient transfer of membrane proteins, and blocking with 5% non-fat milk should be extended to 2 hours to reduce background. If sensitivity remains an issue, consider using chemiluminescent substrates with enhanced sensitivity or implementing immunoprecipitation to concentrate the protein before detection.

How can researchers validate the specificity of a CYP71B23 antibody in plant systems?

Validating CYP71B23 antibody specificity requires a multi-faceted approach. Begin with genetic controls by testing the antibody against protein extracts from confirmed cyp71B23 knockout mutants, which should show absence of the specific band present in wild-type samples. For positive controls, express recombinant CYP71B23 protein (with an orthogonal tag like His or FLAG) in E. coli or yeast systems and confirm detection by both the CYP71B23 antibody and tag-specific antibodies.

Perform peptide competition assays by pre-incubating the antibody with excess immunogenic peptide (if a peptide antibody) before Western blotting - specific signals should be abolished or significantly reduced. To assess potential cross-reactivity with closely related P450 family members, test the antibody against recombinant proteins of the most similar CYP71 family members (e.g., CYP71B6, CYP71B15). Consider developing a CYP71B23-GFP fusion under native promoter for expression in cyp71B23 mutant plants, then confirm co-localization of GFP fluorescence with CYP71B23 antibody immunofluorescence signals. Finally, use mass spectrometry to identify proteins immunoprecipitated by the CYP71B23 antibody to confirm that the primary captured protein is indeed CYP71B23.

What approaches can optimize immunofluorescence experiments using CYP71B23 antibodies for subcellular localization studies?

For optimal immunofluorescence experiments with CYP71B23 antibodies, implement a protocol specifically designed for membrane-associated proteins. Begin with sample preparation by fixing Arabidopsis tissues in 4% paraformaldehyde with 0.1% glutaraldehyde for 2 hours to preserve membrane structures. For permeabilization, use a combination of 0.1% Triton X-100 and 0.05% saponin, which better permeabilizes ER membranes while maintaining structural integrity.

Perform antigen retrieval using citrate buffer (pH 6.0) at 95°C for 10 minutes to unmask potentially hidden epitopes. Block with 3% BSA supplemented with 0.1% cold fish skin gelatin for 3 hours to reduce non-specific binding to membrane components. Use the CYP71B23 antibody at a 1:100 dilution and incubate for 16 hours at 4°C in a humidified chamber. For visualization, implement tyramide signal amplification to enhance detection sensitivity of low-abundance proteins.

Include co-staining with established ER markers (like CNX-RFP) to confirm the expected ER localization pattern similar to other P450 enzymes . For super-resolution imaging, consider structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) to precisely determine CYP71B23 distribution within the ER network and potential co-localization with other metabolic pathway components. Always include appropriate controls such as omission of primary antibody and testing in cyp71B23 knockout tissues.

How might emerging techniques in protein visualization be combined with CYP71B23 antibodies to advance understanding of dynamic metabolon assembly?

Emerging protein visualization techniques can revolutionize our understanding of CYP71B23's role in dynamic metabolon assembly during plant immune responses. Implement lattice light-sheet microscopy with CYP71B23 antibodies conjugated to quantum dots for long-term tracking of protein movements in living cells with minimal phototoxicity. This approach allows visualization of real-time metabolon assembly during pathogen challenge.

Combine this with multi-color super-resolution microscopy such as PALM (photoactivated localization microscopy) to simultaneously track multiple camalexin biosynthetic enzymes labeled with different fluorophores. For even greater precision, implement correlative light and electron microscopy (CLEM) using CYP71B23 antibodies conjugated to gold nanoparticles to visualize metabolon ultrastructure at nanometer resolution.

To study dynamics of protein-protein interactions, apply Förster resonance energy transfer (FRET) sensors incorporating CYP71B23 and interaction partners to monitor association/dissociation events during immune responses. Additionally, consider implementing proximity-dependent biotin identification (BioID) with CYP71B23 as the bait protein to map the entire interactome associated with metabolon formation in living plant cells. These advanced visualization techniques will provide unprecedented insights into the spatial and temporal dynamics of metabolon assembly that cannot be captured through traditional biochemical approaches alone.

What are the prospects for developing monoclonal antibodies against CYP71B23 for enhanced experimental applications?

Developing monoclonal antibodies against CYP71B23 presents significant advantages over the currently available polyclonal antibodies but requires strategic approaches to overcome challenges associated with membrane-bound P450 enzymes. The primary advantage would be exceptional specificity for a single epitope, reducing cross-reactivity with related CYP71 family members that may confound current studies. This would enable more precise protein quantification, especially in comparative studies across different plant stress conditions.

For development, focus on generating immunogens from unique, surface-exposed regions of CYP71B23 identified through structural modeling against crystallized P450 enzymes. Consider using synthetic peptides conjugated to carrier proteins like KLH for regions unique to CYP71B23 compared to other CYP71 enzymes. Alternatively, express recombinant hydrophilic domains of CYP71B23 that maintain native folding while eliminating transmembrane regions that complicate immunization.

To increase success rates, immunize mice with multiple immunogen forms and screen hybridoma clones extensively against both recombinant protein and plant extracts from wild-type versus cyp71B23 knockout plants. Validate selected clones for applications including Western blotting, immunoprecipitation, ChIP assays, and immunofluorescence microscopy. The resulting monoclonal antibodies would enable new experimental approaches including precise stoichiometric analysis of CYP71B23 within metabolons and potentially therapeutic applications targeting homologous P450 enzymes in pathogenic organisms.

How might researchers leverage CYP71B23 antibodies to explore evolutionary conservation of specialized metabolic pathways across plant species?

To investigate evolutionary conservation of specialized metabolic pathways using CYP71B23 antibodies, implement a comprehensive phylogenomic approach. Begin with in silico analysis to identify CYP71B23 orthologs across plant lineages, focusing particularly on species spanning the evolutionary trajectory from basal land plants to advanced angiosperms. Test the cross-reactivity of existing CYP71B23 antibodies against protein extracts from these diverse species, potentially identifying conserved epitopes that indicate functional domains maintained through evolution.

For species where antibody cross-reactivity is confirmed, perform comparative subcellular localization studies to determine if the ER-association pattern is conserved. Additionally, conduct co-immunoprecipitation experiments across species to identify whether protein-protein interaction networks involving CYP71B23 orthologs have been conserved or have diverged during evolution. Complement antibody-based studies with heterologous expression of CYP71B23 orthologs from different species in Arabidopsis cyp71B23 mutants to assess functional complementation.

Analyze metabolic profiles of these transgenic plants to determine if the catalytic function has been conserved despite sequence divergence. This integrated approach will provide insights into how specialized metabolic pathways involving CYP71B23 have evolved, potentially identifying the ancestral function of this enzyme family and how it has been recruited into different biochemical pathways across plant lineages through evolutionary time.

How can CYP71B23 antibodies be applied in studies investigating plant responses to combined biotic and abiotic stresses?

For investigating plant responses to combined stresses, implement a factorial experimental design exposing plants to combinations of biotic stressors (bacterial pathogens, fungi, herbivores) and abiotic stressors (drought, salinity, temperature extremes). Use CYP71B23 antibodies to monitor protein accumulation patterns across these treatment combinations through time-course sampling (0, 12, 24, 48, 72 hours).

Combine Western blot analysis with tissue-specific immunohistochemistry to determine if stress combinations alter not only the expression level but also the tissue distribution of CYP71B23. Implement subcellular fractionation followed by immunoblotting to determine if stress combinations trigger redistribution of CYP71B23 within cellular compartments, potentially indicating metabolon reorganization.

Perform co-immunoprecipitation using CYP71B23 antibodies across stress treatments to identify stress-specific changes in protein interaction networks. Complement protein studies with metabolomics analysis to correlate CYP71B23 expression patterns with changes in specialized metabolite profiles. This integrated approach will reveal how signaling pathway crosstalk during combined stresses affects CYP71B23 regulation and function, providing insights into complex stress adaptation mechanisms in plants similar to those observed for other defense-related enzymes like CYP71A12 .

What methodological considerations are important when using CYP71B23 antibodies in chromatin immunoprecipitation (ChIP) experiments to study transcriptional regulation?

When adapting CYP71B23 antibodies for ChIP experiments, several methodological considerations are crucial for successful outcomes. First, determine if the CYP71B23 antibody recognizes a transcription factor or chromatin-associated protein that regulates CYP71B23 expression, rather than the CYP71B23 enzyme itself (which is not expected to bind DNA directly as a P450 enzyme). If targeting a transcription factor, validate antibody specificity against the recombinant transcription factor and confirm nuclear localization through immunofluorescence microscopy.

For crosslinking, implement a dual crosslinking approach using 1% formaldehyde followed by ethylene glycol bis(succinimidyl succinate) (EGS) to capture more transient protein-DNA interactions. Optimize sonication conditions to generate DNA fragments of 200-500 bp, which is optimal for resolution in ChIP experiments. During immunoprecipitation, include additional washing steps with lithium chloride buffer to reduce background.

For ChIP-qPCR validation, design primers spanning predicted binding sites in the CYP71B23 promoter based on bioinformatic analysis of transcription factor binding motifs. Include negative control regions (gene deserts) and positive control regions (promoters of known target genes) to establish specificity. For genome-wide analysis, implement ChIP-seq with appropriate input controls and biological replicates. Analyze data using peak calling algorithms specifically optimized for transcription factor ChIP-seq data, such as MACS2 with appropriate parameters for plant genomes.

How can researchers effectively use CYP71B23 antibodies to investigate post-translational modifications affecting enzyme function?

To investigate post-translational modifications (PTMs) of CYP71B23, implement a comprehensive strategy combining targeted and untargeted approaches. Begin with immunoprecipitation using CYP71B23 antibodies under native conditions from plants subjected to various biotic and abiotic stresses to capture the enzyme in different modification states. Subject immunoprecipitated protein to mass spectrometry analysis using both bottom-up (peptide level) and top-down (intact protein) proteomics approaches to identify PTMs including phosphorylation, glycosylation, ubiquitination, and sumoylation.

For phosphorylation specifically, perform Western blotting with general phospho-specific antibodies (phospho-serine/threonine/tyrosine) after CYP71B23 immunoprecipitation. Generate site-specific phospho-antibodies against predicted regulatory phosphorylation sites identified through mass spectrometry to monitor specific modification events during stress responses.