CYP94B3 Antibody

What is CYP94B3 and why is it important in plant research?

CYP94B3 is a cytochrome P450 enzyme that functions as a JA-Ile-12-hydroxylase, converting jasmonoyl-L-isoleucine (JA-Ile) to 12-hydroxy-JA-Ile (12OH-JA-Ile) in plants. It plays a crucial role in regulating jasmonate hormone levels through a negative feedback mechanism. The importance of CYP94B3 lies in its ability to attenuate jasmonate responses by catalyzing the hydroxylation of JA-Ile, which reduces its bioactivity in promoting COI1-JAZ receptor complex formation . Studies have shown that CYP94B3 is wound- and jasmonate-responsive, and its activity significantly impacts plant development, male fertility, growth regulation, and defense responses against insect attacks . Researchers interested in plant hormone metabolism, stress responses, or plant immunity would benefit from studying this enzyme and its regulatory mechanisms.

How specific are commercial CYP94B3 antibodies across different plant species?

Commercial CYP94B3 antibodies typically exhibit variable cross-reactivity across plant species depending on sequence conservation. Most available antibodies are developed against Arabidopsis thaliana CYP94B3, which shows high sequence homology with orthologs in closely related species. When working with non-model plants, researchers should verify antibody specificity through pilot experiments using positive controls (such as recombinant CYP94B3 protein) and negative controls (such as protein extracts from cyp94b3 knockout mutants) . Western blotting with decreasing protein concentrations can help establish detection thresholds and specificity. For maximum specificity in experimental systems outside Arabidopsis, researchers may need to validate commercial antibodies or consider custom antibody production using species-specific immunogenic epitopes from the target CYP94B3 protein.

What is the best sample preparation method for detecting CYP94B3 in plant tissues?

The optimal sample preparation method for detecting CYP94B3 depends on both the plant tissue being analyzed and the downstream application. For most applications, microsomal fractions provide better results than crude extracts since CYP94B3 is a membrane-associated cytochrome P450 enzyme. Begin by homogenizing plant tissue (preferably wounded leaves where CYP94B3 expression is induced) in cold extraction buffer containing protease inhibitors and reducing agents (DTT or β-mercaptoethanol) . After initial low-speed centrifugation (10,000g for 15 minutes) to remove debris, ultracentrifuge the supernatant (100,000g for 1 hour) to obtain the microsomal fraction containing membrane proteins. For immunodetection, solubilize the microsomal pellet in a buffer containing a mild detergent such as 1% Triton X-100 or CHAPS. When preparing samples from wounded tissue, timing is critical – optimal detection occurs 1-5 hours post-wounding when CYP94B3 expression peaks in response to increased JA-Ile levels .

How does sample timing affect CYP94B3 detection in wounded plant tissues?

Timing is a critical factor when detecting CYP94B3 in wounded plant tissues due to its wound-inducible expression pattern. According to studies, CYP94B3 expression increases significantly in response to wounding, with expression levels rising within 30 minutes and peaking between 1-5 hours post-wounding . This expression pattern correlates with the accumulation of JA-Ile followed by 12OH-JA-Ile in the tissue. For optimal CYP94B3 protein detection, researchers should collect samples approximately 1-5 hours after wounding, which coincides with the hyperaccumulation of transcripts for JA-responsive genes such as JAZ8 and JAZ10 . Earlier sampling (under 30 minutes) may yield low detection levels since the expression is just beginning to increase, while sampling too late (beyond 12 hours) may result in decreased signals as the wound response attenuates. Additionally, researchers should note that unwounded control tissues typically show minimal CYP94B3 expression and may require more sensitive detection methods.

How can CYP94B3 antibodies be used to study jasmonate signaling dynamics?

CYP94B3 antibodies provide powerful tools for investigating the temporal and spatial dynamics of jasmonate signaling pathways. Researchers can employ immunoprecipitation combined with mass spectrometry (IP-MS) to identify protein complexes associated with CYP94B3 during different stages of the wound response or under various stress conditions. This approach can reveal novel interaction partners that may regulate CYP94B3 activity or localization . For studying the feedback regulation of jasmonate signaling, co-immunoprecipitation experiments using CYP94B3 antibodies alongside antibodies against components of the COI1-JAZ complex can map the physical interactions that modulate signal transduction. Additionally, chromatin immunoprecipitation (ChIP) assays using antibodies against transcription factors (like MYC2) followed by RT-qPCR for the CYP94B3 promoter can elucidate how CYP94B3 expression is regulated at the transcriptional level. Time-course immunofluorescence studies can track the subcellular localization of CYP94B3 in response to wounding, providing insights into compartmentalization of jasmonate metabolism.

What approaches are effective for studying CYP94B3 interactions with other enzymes in the jasmonate metabolism pathway?

To study CYP94B3 interactions with other enzymes in the jasmonate metabolism pathway, researchers can employ multiple complementary approaches. Proximity-dependent biotin identification (BioID) or proximity ligation assays (PLA) using CYP94B3 antibodies can identify proteins that physically interact with CYP94B3 within their native cellular context . Co-immunoprecipitation experiments followed by western blotting can confirm direct interactions between CYP94B3 and candidate proteins like JAR1 (the enzyme responsible for JA-Ile formation) or CYP94C1 (which further oxidizes 12OH-JA-Ile to 12COOH-JA-Ile) . For studying enzyme complexes in planta, bimolecular fluorescence complementation (BiFC) combined with verification using CYP94B3 antibodies provides visualization of interactions in living cells. To investigate the functional consequences of these interactions, researchers can monitor changes in enzyme activity and metabolite levels in wild-type plants versus cyp94b3 mutants using targeted metabolomics approaches. Sequential immunoprecipitation experiments can help determine whether CYP94B3 participates in larger multi-enzyme complexes that might coordinate jasmonate metabolism.

How can immunolocalization techniques be optimized for CYP94B3 detection in different plant tissues?

Optimizing immunolocalization techniques for CYP94B3 detection requires careful consideration of tissue-specific factors and the membrane-associated nature of this cytochrome P450. For paraffin-embedded sections, an extended antigen retrieval step (15-20 minutes) using citrate buffer (pH 6.0) helps expose CYP94B3 epitopes that may be masked during fixation. When using immunofluorescence, researchers should incorporate a membrane permeabilization step with 0.2-0.5% Triton X-100 to facilitate antibody access to the endoplasmic reticulum where CYP94B3 is typically localized . The use of tyramide signal amplification (TSA) can significantly improve detection sensitivity in tissues with lower CYP94B3 expression. For electron microscopy immunogold labeling, optimal dilutions of primary CYP94B3 antibodies (typically 1:50 to 1:200) should be empirically determined for each tissue type. To minimize background in reproductive tissues (where non-specific binding can be problematic), including extended blocking steps (2-3 hours) with 3-5% BSA and normal serum from the secondary antibody host species is recommended. Dual immunolabeling with markers for the endoplasmic reticulum (like calnexin) can confirm the subcellular localization of CYP94B3.

What experimental approaches best demonstrate the functional relationship between CYP94B3 and the COI1-JAZ receptor complex?

To demonstrate the functional relationship between CYP94B3 and the COI1-JAZ receptor complex, researchers should employ a multi-faceted experimental approach. In vitro pull-down assays comparing the ability of JA-Ile versus 12OH-JA-Ile (the product of CYP94B3 activity) to promote COI1-JAZ interactions provide direct biochemical evidence of how CYP94B3 activity affects receptor complex formation . These assays have shown that 12OH-JA-Ile is significantly less effective than JA-Ile in promoting these interactions. Complementary in vivo approaches include comparing JAZ protein stability in wild-type versus cyp94b3 mutant plants using western blotting with antibodies against JAZ proteins (such as JAZ1, JAZ3, or JAZ10) . Researchers can also conduct gene expression analysis for JA-responsive genes in wild-type, cyp94b3 mutant, and CYP94B3-overexpressing plants to correlate CYP94B3 activity with downstream signaling outputs. Chromatin immunoprecipitation (ChIP) assays targeting JAZ-bound genomic regions in these different genetic backgrounds can further elucidate how CYP94B3-mediated JA-Ile catabolism impacts gene regulation. For a systems-level understanding, quantitative phosphoproteomics comparing wild-type and cyp94b3 mutants can identify signaling cascades affected by altered JA-Ile homeostasis.

What controls are essential when using CYP94B3 antibodies for immunoblotting experiments?

When conducting immunoblotting experiments with CYP94B3 antibodies, several essential controls must be included to ensure reliable and interpretable results. Most critically, include protein extracts from cyp94b3 knockout mutant plants as a negative control to confirm antibody specificity and identify any cross-reactivity with related cytochrome P450 enzymes . Conversely, samples from CYP94B3-overexpressing plants serve as positive controls and can help establish the detection limit of the antibody. For loading controls, use antibodies against constitutively expressed membrane proteins (like H+-ATPase) rather than typical soluble protein markers, as these better represent the fraction containing CYP94B3. To verify the molecular weight of detected bands (approximately 57-60 kDa for Arabidopsis CYP94B3), include recombinant CYP94B3 protein when available. For experiments examining wound induction, a time-course sampling is advisable, with unwounded tissue serving as a baseline control. Additionally, testing the antibody against recombinant CYP94B1 and CYP94C1 proteins helps determine potential cross-reactivity with the most closely related family members. The inclusion of peptide competition assays, where the antibody is pre-incubated with excess immunizing peptide, provides further validation of specificity.

What are the optimal fixation and permeabilization protocols for CYP94B3 immunolocalization in plant cells?

The optimal fixation and permeabilization protocols for CYP94B3 immunolocalization must balance preserving antigenicity while maintaining subcellular structure integrity. For light microscopy applications, fix freshly harvested plant tissues in 4% paraformaldehyde in phosphate buffer (pH 7.2) for 2-3 hours at room temperature or overnight at 4°C, as this preserves CYP94B3 epitopes better than glutaraldehyde-based fixatives . Following fixation, wash thoroughly and permeabilize with 0.3-0.5% Triton X-100 for 15-30 minutes to facilitate antibody penetration to the endoplasmic reticulum membrane where CYP94B3 localizes. For electron microscopy, a lower concentration of glutaraldehyde (0.1-0.25%) combined with paraformaldehyde provides better ultrastructural preservation while maintaining reasonable antigenicity. When working with thick tissues like stems or leaves, vacuum infiltration during fixation improves reagent penetration. For optimal antigen retrieval, treat sections with 10mM sodium citrate buffer (pH 6.0) at 95°C for 10-15 minutes before blocking and antibody incubation. The blocking solution should contain 3-5% BSA and 0.1% Tween-20 in PBS, with incubation for at least 1 hour at room temperature to minimize non-specific binding. Test different primary antibody dilutions (ranging from 1:100 to 1:1000) to determine optimal signal-to-noise ratios for each tissue type.

How should researchers quantify CYP94B3 protein levels in comparative studies?

For accurate quantification of CYP94B3 protein levels in comparative studies, researchers should implement a multi-step methodological approach. Begin with careful sample preparation using a standardized protocol that includes protease inhibitors and maintains consistent temperature conditions to prevent degradation of the target protein . For western blotting, load equal amounts of microsomal fraction proteins (20-50 μg per lane) as determined by Bradford or BCA assays. To ensure quantitative linearity, create a standard curve using serial dilutions of a reference sample to confirm that measurements fall within the linear detection range of the antibody and imaging system. Apply normalization using appropriate membrane protein loading controls like H+-ATPase or calnexin rather than cytosolic markers. For image analysis, use software that provides integrated density values from immunoblots, with background subtraction applied consistently across all samples. Biological replicates (minimum n=3) from independent plant samples are essential, and technical replicates can strengthen statistical confidence. For absolute quantification, consider parallel analysis with known quantities of recombinant CYP94B3 protein to establish a calibration curve. When comparing CYP94B3 levels across different genetic backgrounds or treatments, present data as fold-change relative to appropriate controls, and apply suitable statistical tests (such as ANOVA followed by Tukey's test) to determine significance.

What are the key considerations when developing custom CYP94B3 antibodies for specific research applications?

When developing custom CYP94B3 antibodies for specific research applications, researchers must carefully consider several key factors to ensure optimal performance. First, epitope selection is critical—avoid highly conserved regions shared with other CYP94 family members (particularly CYP94B1 and CYP94C1) to minimize cross-reactivity . The N-terminal region and specific loops of CYP94B3 typically offer greater sequence uniqueness compared to the more conserved catalytic domains. For antibodies intended for immunoprecipitation or ChIP applications, target surface-exposed epitopes; for western blotting, linear epitopes often perform better. When designing immunizing peptides, aim for 15-20 amino acids in length with hydrophilicity and predicted antigenicity. Consider producing both polyclonal antibodies (offering higher sensitivity but potentially lower specificity) and monoclonal antibodies (providing higher specificity but potentially more limited epitope recognition). For applications requiring detection of CYP94B3 in its native conformation, immunize with full-length recombinant protein expressed in eukaryotic systems rather than peptides. During antibody production, perform extensive validation using positive controls (recombinant protein, overexpression lines), negative controls (knockout mutants), and closely related family members to assess specificity. For applications requiring higher sensitivity, consider developing nanobodies or recombinant antibody fragments with enhanced tissue penetration properties.

How can researchers address weak or absent CYP94B3 signal in immunoblotting experiments?

When confronting weak or absent CYP94B3 signals in immunoblotting experiments, researchers should systematically troubleshoot several potential issues. First, confirm that CYP94B3 expression has been properly induced in the samples, as basal expression in unwounded tissue is typically very low—collecting tissue 1-5 hours post-wounding significantly increases detection probability . Ensure that proper microsomal fractionation has been performed, as CYP94B3 is a membrane-associated protein that may be lost in crude extracts or cytosolic fractions. Sample preparation should include protease inhibitor cocktails to prevent degradation. For improved protein extraction, try alternative detergents (CHAPS, DDM, or Triton X-100 at 0.5-1%) for membrane solubilization. If signal remains weak, implement signal enhancement strategies such as increased protein loading (50-100 μg), longer primary antibody incubation (overnight at 4°C), higher antibody concentration (1:500 instead of 1:1000), or more sensitive detection methods like chemiluminescence substrate with extended exposure times. Consider using a signal amplification system like biotin-streptavidin or tyramide signal amplification if conventional methods fail. Additionally, check transfer efficiency by staining membranes with Ponceau S after transfer, and optimize transfer conditions for high-molecular-weight membrane proteins (using lower methanol concentration or longer transfer times). For particularly challenging samples, try alternative blocking agents (like 5% BSA instead of milk) that might reduce background while preserving specific binding.

What factors contribute to non-specific binding of CYP94B3 antibodies and how can they be mitigated?

Non-specific binding of CYP94B3 antibodies can arise from multiple factors that require specific mitigation strategies. Cross-reactivity with related cytochrome P450 enzymes, particularly other CYP94 family members (like CYP94B1, CYP94B2, or CYP94C1), represents a major challenge due to sequence homology . To address this, increase antibody specificity by using more stringent washing conditions (0.1-0.2% Tween-20 in TBS/PBS) and optimize blocking conditions (5% BSA or 5% milk with 0.05% Tween-20 for 1-2 hours). For persistent cross-reactivity, consider pre-absorbing the antibody with recombinant proteins of closely related family members or with protein extracts from cyp94b3 knockout plants. Hydrophobic interactions between antibodies and membrane proteins can cause high background in microsomal fractions—mitigate this by adding 0.05-0.1% SDS to washing buffers and reducing primary antibody concentration. Another common issue is epitope masking due to protein-protein interactions or post-translational modifications; mild denaturing conditions during sample preparation (heating at 70°C instead of 95°C) can help maintain epitope accessibility while reducing aggregation of membrane proteins. For immunofluorescence applications where autofluorescence is problematic, treatment with 0.1% sodium borohydride before blocking can reduce background fluorescence. Additionally, using monovalent antibody fragments (Fab) instead of whole IgG molecules can reduce non-specific binding in some applications.

How should researchers interpret and validate unexpected molecular weight patterns in CYP94B3 western blots?

Interpreting and validating unexpected molecular weight patterns in CYP94B3 western blots requires systematic investigation of potential biological and technical explanations. The predicted molecular weight of Arabidopsis CYP94B3 is approximately 57-60 kDa, but researchers may observe multiple bands or bands at unexpected sizes . To validate such observations, first confirm band specificity by comparing patterns between wild-type and cyp94b3 mutant samples—specific bands should be absent or significantly reduced in mutants. For higher-than-expected molecular weight bands, investigate potential post-translational modifications like glycosylation, ubiquitination, or SUMOylation by treating samples with appropriate deglycosylation enzymes or running parallel blots with antibodies against ubiquitin or SUMO. Lower-than-expected molecular weight bands might indicate proteolytic degradation—address this by adding a more comprehensive protease inhibitor cocktail during extraction and keeping samples consistently cold. To distinguish between splice variants and degradation products, perform RT-PCR to detect alternative transcripts, then verify if these correspond to the observed protein bands. For particularly challenging interpretations, immunoprecipitate the protein using the CYP94B3 antibody followed by mass spectrometry analysis to definitively identify the observed species. Additionally, testing multiple antibodies targeting different epitopes of CYP94B3 can help confirm whether unexpected bands represent genuine CYP94B3-related proteins or non-specific interactions.

What strategies help overcome tissue-specific challenges when detecting CYP94B3 in different plant organs?

Different plant organs present unique challenges for CYP94B3 detection that require tissue-specific optimization strategies. In leaves, high chlorophyll content and phenolic compounds can interfere with protein extraction and antibody binding—incorporate PVPP (polyvinylpolypyrrolidone) at 2-5% (w/v) during extraction to remove phenolics, and use higher concentrations of reducing agents (5-10 mM DTT) to mitigate oxidation . For reproductive tissues like flowers and developing seeds, high levels of storage proteins and secondary metabolites necessitate modified extraction buffers with additional detergents (0.5-1% NP-40 along with 0.5% Triton X-100) and extended washing steps during immunoprecipitation or immunolocalization. Root tissues generally have lower CYP94B3 expression levels, requiring more sensitive detection methods like enhanced chemiluminescence or signal amplification systems. When working with stems or woody tissues, mechanical disruption efficiency becomes critical—use cryogenic grinding with liquid nitrogen followed by extended extraction times (30-60 minutes) with agitation. For immunohistochemistry in thick or waxy tissues, extended permeabilization (up to 1 hour with 0.5% Triton X-100) and longer primary antibody incubation times (overnight at 4°C) improve penetration and signal development. To address autofluorescence in lignified tissues during immunofluorescence microscopy, pretreat sections with 0.1% sodium borohydride followed by 0.5% Sudan Black B in 70% ethanol before immunolabeling procedures.

How can CYP94B3 antibodies contribute to studies on plant-pathogen and plant-herbivore interactions?

CYP94B3 antibodies offer powerful tools for investigating the molecular dynamics of plant defense responses in plant-pathogen and plant-herbivore interactions. As CYP94B3 regulates jasmonate signaling, which is central to plant immunity, researchers can use these antibodies to track changes in CYP94B3 protein abundance and localization during infection or herbivory events . Immunohistochemistry with CYP94B3 antibodies can reveal spatial patterns of jasmonate metabolism at infection sites or along herbivore feeding tracks, providing insights into the localization of defense responses. For studying systemic acquired resistance, researchers can compare CYP94B3 levels in local and distal tissues using quantitative immunoblotting. Co-immunoprecipitation experiments with CYP94B3 antibodies followed by mass spectrometry can identify pathogen-induced changes in CYP94B3 interaction partners that may explain altered hormone metabolism during defense responses. Time-course studies tracking CYP94B3 abundance after pathogen recognition can reveal how quickly plants mobilize jasmonate catabolism machinery to fine-tune defense responses. Additionally, comparative studies of CYP94B3 protein levels in resistant versus susceptible plant varieties may identify correlations between jasmonate metabolism efficiency and defense outcomes. These approaches can help develop crop protection strategies by revealing how pathogens or herbivores might manipulate jasmonate metabolism to suppress plant defenses.

What role could CYP94B3 antibodies play in understanding cross-talk between jasmonate and other hormone signaling pathways?

CYP94B3 antibodies can serve as invaluable tools for unraveling the complex cross-talk between jasmonate and other hormone signaling pathways in plants. By employing co-immunoprecipitation with CYP94B3 antibodies followed by mass spectrometry, researchers can identify proteins that interact with CYP94B3 under different hormonal treatments (such as auxin, ethylene, abscisic acid, or salicylic acid), potentially revealing direct molecular connections between these pathways . Chromatin immunoprecipitation experiments using antibodies against transcription factors from various hormone pathways, followed by qPCR analysis of the CYP94B3 promoter region, can demonstrate how other hormones might directly regulate CYP94B3 expression. For examining post-translational regulation, phospho-specific antibodies can be developed to detect potential phosphorylation of CYP94B3 by kinases activated in response to other hormones. Comparative quantitative immunoblotting of CYP94B3 in wild-type plants versus mutants impaired in other hormone pathways can reveal regulatory influences between signaling networks. Dual immunolocalization studies using CYP94B3 antibodies alongside markers for other hormone biosynthesis or signaling components can identify spatial relationships between these pathways within cellular compartments or across tissues. Such approaches may ultimately help explain phenomena like hormone-mediated growth-defense trade-offs and could lead to strategic manipulation of these pathways for improved crop resilience.

How can quantitative proteomics approaches incorporating CYP94B3 antibodies enhance our understanding of jasmonate metabolism?

Quantitative proteomics approaches incorporating CYP94B3 antibodies can significantly advance our understanding of jasmonate metabolism through several sophisticated techniques. Immunoprecipitation using CYP94B3 antibodies coupled with mass spectrometry (IP-MS) enables identification of protein complexes and interactors that may regulate CYP94B3 activity or localization within cellular compartments . This approach can be extended to comparative analyses across different stress conditions or developmental stages to map dynamic changes in the CYP94B3 interactome. Selective Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM) mass spectrometry using immunopurified CYP94B3 can provide absolute quantification of this enzyme across different tissues or treatments with high sensitivity. For studying post-translational modifications that might regulate CYP94B3 activity, researchers can use immunoprecipitated CYP94B3 for phosphoproteomic, glycoproteomic, or ubiquitylation site analyses. To examine CYP94B3 within the broader context of jasmonate metabolic enzymes, sequential immunoprecipitation with antibodies against CYP94B3 followed by antibodies against other pathway enzymes (like JAR1 or CYP94C1) can reveal whether these proteins form functional metabolons. Proximity-dependent labeling methods such as BioID or APEX2 fused to CYP94B3 can identify proximal proteins in living cells, while subsequent validation with antibody-based techniques can confirm these interactions. These approaches collectively provide a systems-level view of how CYP94B3 functions within the complex network of jasmonate metabolism.

What future developments in antibody technology could improve CYP94B3 detection and functional studies?

Future developments in antibody technology promise to enhance CYP94B3 detection and functional studies through several innovative approaches. The development of nanobodies (single-domain antibodies derived from camelids) against CYP94B3 offers advantages including smaller size for better tissue penetration, higher stability, and the potential for direct expression in planta as intrabodies to track or modulate CYP94B3 function in real-time . Proximity-dependent labeling techniques could advance significantly through antibody-enzyme fusion proteins, where CYP94B3 antibodies are directly conjugated to enzymes like APEX2 or TurboID for in situ identification of interaction partners with temporal precision. For enhanced specificity, the development of recombinant antibodies through phage display technology could generate CYP94B3-specific binding proteins with minimal cross-reactivity to other CYP94 family members. Spatiotemporal dynamics of CYP94B3 could be better visualized using optically controlled antibody fragments that can be activated with specific wavelengths of light, enabling precise tracking of CYP94B3 localization during stress responses. Mass cytometry (CyTOF) using metal-conjugated CYP94B3 antibodies could allow for simultaneous detection of multiple proteins in the jasmonate pathway with minimal spectral overlap. Additionally, the development of antibodies specifically recognizing particular post-translational modifications of CYP94B3 (such as phosphorylation, ubiquitination, or glycosylation) would provide invaluable tools for investigating regulatory mechanisms controlling this enzyme's activity and stability in response to environmental stimuli.

What are the most significant recent advances in CYP94B3 antibody applications for plant hormone research?

Recent advances in CYP94B3 antibody applications have significantly expanded our understanding of jasmonate metabolism and signaling in plants. One of the most notable developments has been the application of CYP94B3 antibodies in chromatin immunoprecipitation sequencing (ChIP-seq) studies to identify transcription factors that directly regulate CYP94B3 expression during stress responses . This has revealed how plants coordinate jasmonate catabolism with broader stress response networks. Another significant advance involves the use of proximity-dependent labeling methods like BioID or APEX2, where CYP94B3 antibodies help validate identified interaction partners, providing unprecedented insights into the dynamic protein complexes that regulate jasmonate metabolism. The application of super-resolution microscopy techniques (such as STORM or PALM) with CYP94B3 antibodies has revealed nanoscale spatial organization of jasmonate metabolic enzymes, suggesting the existence of metabolons that enhance pathway efficiency. Protein correlation profiling using CYP94B3 antibodies has mapped co-regulated proteins across developmental stages and stress conditions, identifying new components of jasmonate signaling networks. Additionally, the development of highly specific monoclonal antibodies against CYP94B3 has enabled precise quantification of this enzyme in crop species, facilitating comparative studies of jasmonate metabolism in agriculturally important plants. These advances collectively provide powerful tools for dissecting the molecular mechanisms of hormone signaling and may ultimately contribute to engineering improved stress resistance in crops.