CYP71B4 Antibody

What is CYP71B4 and what biological functions does it serve in plants?

CYP71B4 is a cytochrome P450 monooxygenase enzyme belonging to the CYP71 family, which constitutes one of the largest subfamilies of plant P450 enzymes. The enzyme is encoded in Arabidopsis thaliana and is classified under the broader category of cytochrome P450 monooxygenases that typically catalyze various oxidative reactions in plant metabolism. While specific functions of CYP71B4 are still being elucidated, cytochrome P450 enzymes in this family generally play important roles in biosynthesis of secondary metabolites, plant defense responses, and hormone metabolism. Research indicates that many CYP71 family members participate in tryptophan metabolism and synthesis of defensive compounds, similar to the related enzyme CYP71A12 which is involved in pathogen-triggered tryptophan metabolism . CYP71B4, like other cytochrome P450 enzymes, likely contributes to the plant's metabolic versatility and stress adaptation mechanisms through its enzymatic activities.

What are the key characteristics of commercially available CYP71B4 antibodies?

Commercial CYP71B4 antibodies, such as those available from suppliers like CUSABIO Technology LLC, are typically rabbit polyclonal antibodies raised against recombinant Arabidopsis thaliana CYP71B4 protein . These antibodies are designed to specifically recognize plant antigens and have been validated for various applications including ELISA, Western blot, and other immunoassay techniques . The antibodies are commonly supplied in a liquid format with preservatives such as Proclin 300 (0.03%) and constituents including glycerol (50%) and phosphate-buffered saline (0.01M PBS, pH 7.4) . Unlike monoclonal antibodies, these polyclonal antibodies recognize multiple epitopes on the CYP71B4 protein, potentially offering greater sensitivity in detecting native protein. Commercial preparations often include supplementary components such as recombinant immunogen protein/peptide as a positive control and pre-immune serum for comparison purposes, enhancing experimental validation capabilities .

What are the optimal protocols for using CYP71B4 antibodies in Western blot applications?

For optimal Western blot applications using CYP71B4 antibodies, researchers should begin with proper sample preparation from plant tissues, particularly Arabidopsis thaliana, where this protein is natively expressed. Extraction should be performed using a buffer containing protease inhibitors to prevent protein degradation during the isolation process. Based on protocols used for similar plant proteins, a standard SDS-PAGE setup with 10-12% acrylamide gels is recommended for proper separation of CYP71B4, which has an approximate molecular weight of 55-60 kDa. After electrophoresis, proteins should be transferred to a PVDF or nitrocellulose membrane using standard transfer conditions (typically 100V for 1 hour or 30V overnight at 4°C). Blocking should be performed with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature . The CYP71B4 primary antibody should be diluted according to manufacturer recommendations (typically 1:1000 to 1:2000) in blocking buffer and incubated overnight at 4°C . Following at least three 10-minute washes with TBST, an appropriate HRP-conjugated secondary antibody against rabbit IgG should be applied at a 1:5000 to 1:10000 dilution for 1 hour at room temperature.

What techniques are effective for immunolocalization of CYP71B4 in plant tissues?

Effective immunolocalization of CYP71B4 in plant tissues requires careful sample preparation and optimization of antibody conditions. Begin by fixing fresh plant tissues (preferably from Arabidopsis thaliana) in 4% paraformaldehyde in PBS for 2-4 hours at room temperature, followed by embedding in paraffin or resin. For paraffin embedding, sections should be cut at 5-10 μm thickness, deparaffinized with xylene, and rehydrated through an ethanol gradient. Antigen retrieval is often necessary and can be performed using citrate buffer (pH 6.0) at 95°C for 20 minutes. For immunofluorescence, blocking should be conducted with 2-3% BSA in PBS containing 0.1% Triton X-100 for 1 hour at room temperature. The CYP71B4 antibody should be applied at dilutions ranging from 1:100 to 1:500 in blocking buffer and incubated overnight at 4°C . After washing with PBS-T (at least three 10-minute washes), apply fluorophore-conjugated secondary antibodies (like Alexa Fluor 488 anti-rabbit) at 1:200 to 1:500 dilution for 1-2 hours at room temperature in darkness. Counterstaining with DAPI (1 μg/ml) for 5 minutes can help visualize nuclei. Confocal microscopy techniques similar to those used for localization of other plant proteins like ACBP6 can be employed for high-resolution imaging of CYP71B4 subcellular distribution .

How can researchers verify the specificity of CYP71B4 antibodies in their experimental systems?

To verify antibody specificity, researchers should implement multiple complementary approaches. First, perform Western blot analysis using purified recombinant CYP71B4 protein alongside plant extracts to confirm that the antibody recognizes a band of the expected molecular weight (approximately 55-60 kDa). The recombinant immunogen protein provided with some commercial antibodies serves as an excellent positive control for this purpose . Second, include a negative control by testing the antibody against extracts from cyp71b4 knockout mutant plants, where absence of the target band would confirm specificity. Pre-immune serum controls, often provided with commercial antibodies, should also be used to assess non-specific binding . Third, peptide competition assays can be conducted by pre-incubating the antibody with excess CYP71B4 antigenic peptide before application to the sample; successful competition should eliminate specific signals. Fourth, immunoprecipitation followed by mass spectrometry analysis can provide definitive identification of the precipitated proteins, confirming that the antibody is indeed capturing CYP71B4. Finally, cross-reactivity against related cytochrome P450 enzymes should be evaluated, particularly other members of the CYP71 family, to ensure that the observed signals are specifically attributable to CYP71B4.

What are common causes of non-specific binding with CYP71B4 antibodies and how can they be minimized?

Non-specific binding with CYP71B4 antibodies can arise from several sources and requires systematic troubleshooting. One common cause is insufficient blocking, which can be addressed by increasing blocking time (up to 2 hours) or concentration (3-5% BSA or non-fat dry milk), or by using alternative blocking agents like casein or commercial blocking buffers that may be more effective with particular antibodies. Another frequent issue is excessively high antibody concentration, which can be resolved by performing careful titration experiments to determine the optimal dilution that maximizes specific signal while minimizing background. Cross-reactivity with related cytochrome P450 enzymes, particularly those within the CYP71 family that share sequence homology, can be problematic; this issue can be mitigated by using affinity-purified antibodies or by pre-absorbing the antibody with recombinant proteins of closely related family members. High endogenous peroxidase activity in plant tissues can generate false positive signals in protocols using HRP-conjugated secondary antibodies; this can be addressed by including a peroxidase quenching step (0.3% H₂O₂ in methanol for 30 minutes) before antibody application. Lastly, detergent concentration in wash buffers may be insufficient to remove weakly bound antibodies; increasing Tween-20 concentration to 0.1-0.3% in wash buffers can help reduce this type of background.

How can CYP71B4 antibodies be utilized in co-immunoprecipitation studies to identify protein interaction partners?

Co-immunoprecipitation (Co-IP) using CYP71B4 antibodies provides a powerful approach for identifying protein interaction partners that may illuminate the enzyme's biological functions and regulatory networks. To optimize Co-IP protocols, researchers should begin with gentle lysis conditions using non-denaturing buffers (typically containing 0.5-1% NP-40 or Triton X-100, 150 mM NaCl, 50 mM Tris-HCl pH 7.5, and protease inhibitors) to preserve protein-protein interactions. Pre-clearing the lysate with protein A/G beads for 1 hour at 4°C helps reduce non-specific binding. CYP71B4 antibodies should be conjugated to protein A/G magnetic beads or agarose beads at optimal ratios (typically 2-5 μg antibody per 20-50 μl bead slurry) before incubation with the pre-cleared lysate overnight at 4°C with gentle rotation . After stringent washing (at least 4-5 washes with lysis buffer), eluted proteins can be analyzed by SDS-PAGE followed by silver staining or Western blotting for suspected interaction partners. For unbiased discovery of novel interaction partners, mass spectrometry analysis of the co-immunoprecipitated proteins is recommended. Control experiments should include parallel Co-IPs with pre-immune serum or IgG from the same species, as well as lysates from cyp71b4 knockout plants to identify non-specific interactions. Cross-linking approaches using membrane-permeable cross-linkers like DSP or formaldehyde before cell lysis can help capture transient or weak interactions that might otherwise be lost during the procedure.

What strategies can be employed to study CYP71B4 expression patterns under various stress conditions?

Comprehensive analysis of CYP71B4 expression patterns under various stress conditions requires a multi-faceted approach combining protein-level detection with transcriptomic analysis. At the protein level, quantitative Western blot analysis using CYP71B4 antibodies can be performed on plant samples subjected to different stresses (biotic, abiotic, hormonal treatments) . Researchers should establish a time-course experiment exposing plants to stressors for varying durations (e.g., 0, 6, 12, 24, 48, and 72 hours) to capture both early responses and sustained changes in expression patterns. Protein extraction should be optimized to ensure consistent recovery across all conditions, with equal loading verified by parallel detection of housekeeping proteins like actin or tubulin. Immunohistochemistry or immunofluorescence can reveal tissue-specific changes in CYP71B4 expression and subcellular localization in response to stress conditions. These protein-level analyses should be complemented with transcriptomic approaches such as qRT-PCR to measure CYP71B4 mRNA levels, providing insights into transcriptional regulation. For broader context, RNA-seq analysis can position CYP71B4 expression changes within global transcriptional networks, potentially identifying co-regulated genes. Reporter gene constructs (e.g., CYP71B4 promoter::GUS or CYP71B4 promoter::LUC) can provide spatiotemporal visualization of promoter activity in transgenic plants under different stress conditions, similar to approaches used for studying cold-responsive gene expression .

How can CRISPR-Cas9 gene editing be integrated with CYP71B4 antibody-based detection to study enzyme function?

Integrating CRISPR-Cas9 gene editing with CYP71B4 antibody-based detection creates a powerful system for dissecting enzyme function through precise genetic manipulation coupled with protein expression analysis. Researchers should first design specific guide RNAs targeting different regions of the CYP71B4 gene, including the catalytic domain, substrate binding sites, and potential regulatory regions, to generate a series of knockout and domain-specific mutant plants. For knockouts, guide RNAs should target early exons to ensure complete loss of functional protein. CRISPR-Cas9 can also be used to introduce precise mutations that alter specific amino acids hypothesized to be critical for enzyme function or to add epitope tags for enhanced detection. After generating and confirming edited lines through sequencing, CYP71B4 antibodies can be used to verify protein expression changes by Western blot, with knockout lines serving as negative controls to confirm antibody specificity . Comparative phenotypic analysis between wild-type and edited plants under various conditions can reveal functions associated with CYP71B4, while metabolomic profiling can identify specific metabolic pathways affected by the genetic modifications. Complementation experiments, where the wild-type CYP71B4 or mutant variants are reintroduced into knockout backgrounds, can confirm that observed phenotypes are specifically due to CYP71B4 function rather than off-target effects. Antibody-based immunoprecipitation followed by mass spectrometry analysis can further identify changes in protein interaction networks resulting from specific CRISPR-induced modifications.

How can CYP71B4 antibodies be utilized in conjunction with mass spectrometry for comprehensive protein characterization?

The integration of CYP71B4 antibodies with mass spectrometry techniques offers comprehensive characterization of protein abundance, post-translational modifications, and interaction networks. Immunoprecipitation using CYP71B4 antibodies can first be employed to enrich the target protein from complex plant extracts before mass spectrometric analysis . For identification and quantification, the immunoprecipitated proteins should undergo tryptic digestion followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) approaches can be developed for targeted quantification of specific CYP71B4 peptides, enabling precise measurement of protein abundance across different samples or conditions. To characterize post-translational modifications (PTMs), enriched CYP71B4 can be analyzed using electron transfer dissociation (ETD) or higher-energy collisional dissociation (HCD) fragmentation methods, which are particularly effective for identifying phosphorylation, glycosylation, or other modifications that may regulate enzyme activity. Crosslinking mass spectrometry (XL-MS) can be applied to immunoprecipitated complexes to map protein-protein interaction interfaces at amino acid resolution, providing structural insights into CYP71B4 complexes. For even more detailed characterization, top-down proteomics approaches analyzing intact CYP71B4 can reveal proteoforms with different combinations of PTMs that might be missed in bottom-up approaches. Integration of these mass spectrometry data with molecular modeling can further elucidate structure-function relationships of CYP71B4.

What approaches can be used to study the role of CYP71B4 in plant metabolic networks?

Elucidating the role of CYP71B4 in plant metabolic networks requires an integrated systems biology approach combining genetic manipulation, metabolomics, and protein detection. Researchers should generate both knockout and overexpression lines of CYP71B4 in Arabidopsis thaliana as experimental systems. CYP71B4 antibodies can be used to confirm protein absence in knockout lines and quantify expression levels in overexpression lines via Western blot analysis . Untargeted metabolomics using liquid or gas chromatography coupled with mass spectrometry (LC-MS or GC-MS) should be performed on these genetically modified plants compared to wild-type controls, enabling identification of metabolites whose levels are altered by CYP71B4 manipulation. Stable isotope labeling experiments using 13C or 15N-labeled precursors can track metabolic flux through pathways potentially involving CYP71B4, with time-course sampling revealing the kinetics of substrate conversion and product formation. Network analysis of co-regulated genes identified through transcriptomics, combined with metabolite correlation networks, can position CYP71B4 within broader biochemical pathways. Enzyme assays using recombinant CYP71B4 with potential substrates identified through metabolomics can confirm direct enzymatic activities. Similar approaches have been successfully used to characterize the involvement of CYP71A12 in pathogen-triggered tryptophan metabolism and the production of defense compounds like indole-3-carboxylic acid (ICA) .

How can antibody-based techniques be combined with imaging technologies to visualize CYP71B4 dynamics in living plant cells?

Combining antibody-based techniques with advanced imaging technologies enables visualization of CYP71B4 dynamics in living plant cells, providing insights into its subcellular localization, trafficking, and responses to environmental stimuli. While direct antibody application in living cells is challenging due to cell membrane impermeability, several innovative approaches can overcome this limitation. For fixed-cell high-resolution imaging, researchers should use CYP71B4 antibodies in immunofluorescence protocols coupled with super-resolution microscopy techniques such as structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, or stochastic optical reconstruction microscopy (STORM) to achieve nanoscale resolution of protein localization . For live-cell imaging, genetic fusion approaches can be employed where CYP71B4 is fused to fluorescent proteins (e.g., GFP, mCherry) and expressed in transgenic plants, similar to approaches used for studying ACBP6 subcellular localization . The authenticity of fusion protein localization can be validated by comparing fixed-cell immunofluorescence using CYP71B4 antibodies with the fluorescent signal from fusion constructs. Fluorescence recovery after photobleaching (FRAP) or photoactivatable fluorescent proteins can reveal protein mobility and trafficking patterns. For protein-protein interactions in living cells, bimolecular fluorescence complementation (BiFC) or Förster resonance energy transfer (FRET) techniques can be employed, with CYP71B4 antibodies used in parallel co-immunoprecipitation experiments to confirm interactions observed in imaging studies.

How can researchers address contradictory findings when comparing CYP71B4 protein levels with gene expression data?

Discrepancies between CYP71B4 protein levels detected by antibody-based methods and corresponding mRNA expression represent a common challenge that requires systematic investigation of post-transcriptional and post-translational regulatory mechanisms. Researchers should first validate both measurement approaches: for protein quantification, use multiple antibody-based techniques (Western blot, ELISA, immunohistochemistry) with appropriate controls to confirm specificity ; for mRNA quantification, design multiple primer pairs targeting different regions of the CYP71B4 transcript and validate by sequencing amplicons. Time-course experiments can reveal temporal dynamics, as protein levels often lag behind mRNA changes due to translation time and protein half-life differences. Discrepancies may indicate post-transcriptional regulation such as microRNA-mediated suppression or differential mRNA stability, which can be investigated using RNA stability assays with transcription inhibitors like actinomycin D. Post-translational mechanisms including protein degradation rates should be examined using cycloheximide chase assays or by measuring ubiquitination levels of immunoprecipitated CYP71B4. Alternative splicing of CYP71B4 mRNA could generate protein isoforms that might not be equally detected by the antibody; this possibility can be investigated using isoform-specific primers in RT-PCR or by mass spectrometry analysis of the protein. Translational efficiency can be assessed through polysome profiling to determine if CYP71B4 mRNA association with ribosomes correlates with protein levels.

What considerations are important when comparing results from different detection methods using CYP71B4 antibodies?

When comparing results from different detection methods using CYP71B4 antibodies, researchers must account for several technical factors that can influence data interpretation. Each detection method (Western blot, ELISA, immunofluorescence, flow cytometry) has distinct sensitivity thresholds and dynamic ranges; Western blotting may detect denatured epitopes not accessible in native-state ELISA or immunofluorescence techniques . Sample preparation differences significantly impact results: protein extraction methods vary in efficiency across subcellular compartments, potentially biasing detection toward certain protein pools. The antibody concentration should be optimized separately for each technique, as excess antibody may cause non-specific binding in immunofluorescence while being acceptable in Western blots where size separation helps confirm specificity. Detection systems vary in sensitivity, with chemiluminescence typically offering greater sensitivity than colorimetric methods in Western blots, while amplification steps in techniques like tyramide signal amplification can dramatically increase immunofluorescence signals. Quantitative comparisons across methods should include calibration curves using purified recombinant CYP71B4 protein at known concentrations to establish absolute quantification references . When reporting results, researchers should clearly specify all methodological details including antibody source, catalog number, and dilution; detection method specifications; and quantification approaches. Multi-method validation, where the same samples are analyzed by different techniques, provides the strongest evidence for consistent protein detection and can highlight method-specific biases that should be considered during data interpretation.

How might emerging antibody technologies enhance CYP71B4 research in the coming years?

Emerging antibody technologies hold substantial promise for advancing CYP71B4 research through improvements in specificity, sensitivity, and application versatility. Nanobodies—single-domain antibody fragments derived from camelid antibodies—offer advantages of smaller size (approximately 15 kDa compared to 150 kDa for conventional antibodies), enabling better penetration into dense tissues and access to epitopes in protein complexes that might be inaccessible to conventional antibodies. These could be especially valuable for studying CYP71B4 in intact plant structures. Recombinant antibody engineering technologies will allow the development of highly specific monoclonal antibodies against defined CYP71B4 epitopes, potentially distinguishing between closely related cytochrome P450 family members with greater precision than current polyclonal options . Antibody fragments like Fab or scFv can be generated with enhanced tissue penetration properties for immunohistochemistry applications. Proximity labeling approaches such as APEX2 or TurboID fused to anti-CYP71B4 antibodies could enable identification of proteins in close proximity to CYP71B4 in their native cellular environment. Advances in site-specific conjugation chemistry will allow precise attachment of fluorophores, enzymes, or other functional groups to antibodies without compromising binding characteristics. Multi-specific antibodies capable of simultaneously recognizing CYP71B4 and another protein of interest could facilitate co-localization studies with simplified protocols. Additionally, the development of renewable recombinant antibodies with precisely defined production parameters will address batch-to-batch variation issues that currently complicate long-term studies using conventional polyclonal antibodies.

What are promising approaches for studying the role of CYP71B4 in plant-pathogen interactions?

Investigating CYP71B4's role in plant-pathogen interactions requires multidisciplinary approaches combining molecular, cellular, and systems biology techniques. Researchers should establish infection models using well-characterized plant pathogens with different infection strategies (e.g., biotrophic, necrotrophic, and hemibiotrophic pathogens) to determine if CYP71B4 expression is differentially regulated during specific types of pathogen challenges. Time-course experiments monitoring CYP71B4 protein levels using quantitative Western blot analysis during pathogen infection can reveal temporal dynamics of enzyme involvement in defense responses . Spatial regulation can be examined through immunohistochemistry to determine if CYP71B4 accumulates at infection sites or in specific tissues during pathogen challenge. Genetic approaches using CYP71B4 knockout and overexpression lines should be phenotypically characterized for altered susceptibility or resistance to pathogens. Metabolomic analysis of these genetically modified plants during infection can identify defense-related compounds whose production depends on CYP71B4 activity, similar to studies showing CYP71A12's involvement in the biosynthesis of indole-3-carboxylic acid (ICA) during pathogen responses . Investigation of potential protein-protein interactions between CYP71B4 and known defense signaling components using co-immunoprecipitation with CYP71B4 antibodies followed by mass spectrometry could reveal integration points with established defense pathways. Cell-specific expression analysis using fluorescent reporter constructs driven by the CYP71B4 promoter, validated by immunolocalization with CYP71B4 antibodies, can provide insights into tissue-specific roles in defense.

How can computational modeling be integrated with antibody-based experimental data to predict CYP71B4 function?

Integration of computational modeling with antibody-based experimental data offers powerful approaches for predicting CYP71B4 function through multi-scale analysis from protein structure to systems-level interactions. Researchers should begin with homology modeling of CYP71B4 based on crystallized plant cytochrome P450 structures, with model validation performed using experimental data such as site-directed mutagenesis of predicted catalytic residues followed by antibody-based detection of expression and activity changes . Molecular docking simulations can predict potential substrates by virtual screening of plant metabolites against the modeled active site, with predictions verified experimentally through enzyme assays and metabolite profiling of CYP71B4 knockout plants. Quantitative systems pharmacology approaches can integrate antibody-derived protein abundance data with enzyme kinetic parameters to model metabolic flux through pathways involving CYP71B4. Network modeling using protein interaction data from co-immunoprecipitation experiments with CYP71B4 antibodies can place the enzyme within broader signaling and metabolic networks. Machine learning algorithms can be trained on multi-omics datasets incorporating antibody-based protein quantification, transcriptomics, and metabolomics data from various experimental conditions to predict CYP71B4 involvement in specific biological processes or responses to environmental stimuli. Molecular dynamics simulations can provide insights into conformational changes that might affect enzyme activity or protein-protein interactions, with predictions tested experimentally through antibody-based techniques like hydrogen-deuterium exchange mass spectrometry. This iterative process of computational prediction and experimental validation using antibody-based methods creates a virtuous cycle for continually refining understanding of CYP71B4 function.