CYP94B1 Antibody

What is CYP94B1 and how does it function in plant biology?

CYP94B1 belongs to the cytochrome P450 family of enzymes that play critical roles in plant hormone metabolism. Similar to its paralog CYP94B3, it likely participates in the catabolism and inactivation of jasmonoyl-L-isoleucine (JA-Ile), a key plant hormone involved in stress responses and development. CYP94B1 acts through hydroxylation reactions that modify hormonal compounds, effectively regulating their biological activity and contributing to hormone homeostasis in plants .

How does CYP94B1 relate structurally and functionally to other CYP94 family members?

CYP94B1 shares significant structural homology with other CYP94 family members, particularly CYP94B3 and CYP94C1, which are all involved in jasmonate metabolism. While CYP94B3 primarily hydroxylates JA-Ile to form 12-hydroxy-JA-Ile (12OH-JA-Ile), CYP94B1 may exhibit overlapping substrate specificity with potentially distinct regiospecificity or catalytic efficiency. These enzymes collectively form a metabolic network that fine-tunes jasmonate signaling through sequential oxidation reactions .

What experimental approaches have been validated for studying CYP94B1 expression and function?

Validated approaches include:

• T-DNA insertion mutant analysis for gene function studies

• RT-qPCR for expression analysis under different stress conditions

• Heterologous expression in yeast systems for in vitro enzyme assays

• In vitro hydroxylation assays using microsomal preparations

• Metabolite profiling through LC-MS/MS to track hormone catabolites

• Phenotypic analysis of knockout/overexpression lines

What are the critical considerations when selecting a CYP94B1 antibody for research applications?

When selecting a CYP94B1 antibody, researchers should consider:

• Antibody specificity, particularly cross-reactivity with other CYP94 family members

• Validated applications (WB, IHC, IP, ELISA)

• Host species and clonality (monoclonal vs. polyclonal)

• Immunogen design and epitope location

• Validation data in plant systems

• Lot-to-lot consistency documentation

• Published citations demonstrating successful application

How can I rigorously validate the specificity of a CYP94B1 antibody?

A comprehensive validation approach includes:

• Western blot analysis comparing wild-type and cyp94b1 knockout tissues

• Peptide competition assays using the immunogenic peptide

• Immunoprecipitation followed by mass spectrometry identification

• Parallel testing with multiple antibodies recognizing different epitopes

• Heterologous expression of CYP94B1 in a null background as positive control

• Immunohistochemical localization matching known expression patterns

What protocols effectively minimize non-specific binding when using CYP94B1 antibodies?

To minimize non-specific binding:

• Optimize blocking conditions (5% BSA or milk in TBS-T for 1-2 hours)

• Include detergents like 0.1% Tween-20 in washing buffers

• Pre-adsorb antibody with plant extracts from cyp94b1 knockout tissues

• Implement stringent washing procedures (at least 3x15 minutes)

• Titrate primary antibody concentration (typically 1:500-1:1500 for WB)

• Use highly purified secondary antibodies with minimal cross-reactivity

• Consider signal amplification methods for low-abundance targets

What is the optimal Western blot protocol for detecting CYP94B1 in plant tissues?

Optimized Western Blot Protocol for CYP94B1:

This protocol can be adjusted based on sample type and antibody specifications .

How can I effectively use CYP94B1 antibodies for immunolocalization studies in plant tissues?

For effective immunolocalization:

• Fix tissues in 4% paraformaldehyde for optimal antigen preservation

• Perform antigen retrieval using either TE buffer (pH 9.0) or citrate buffer (pH 6.0)

• Block with 2-5% normal serum from the same species as the secondary antibody

• Apply primary CYP94B1 antibody at 1:20-1:200 dilution (optimize for specific antibody)

• Include appropriate controls: no primary antibody, pre-immune serum, and cyp94b1 knockout tissue

• Use fluorescent or enzymatic detection systems with minimal background

• Counter-stain with organelle markers to confirm subcellular localization

What approaches can be used to develop a quantitative immunoassay for measuring CYP94B1 levels?

Researchers can develop quantitative immunoassays using:

• ELISA systems with purified recombinant CYP94B1 as standards

• Piezoimmunosensors using scFv antibody fragments for enhanced sensitivity

• Quantitative Western blot with infrared fluorescent detection systems

• Bead-based multiplexed immunoassays for simultaneous detection of multiple CYP enzymes

• Sandwich ELISA configurations to improve specificity in complex samples

These approaches can be adapted from established methods for other CYP family members .

What are the most common technical challenges when working with CYP94B1 antibodies and how can they be addressed?

These solutions should be systematically tested to identify optimal conditions for specific experimental contexts .

How can I address cross-reactivity issues between CYP94B1 and other closely related CYP family members?

To address cross-reactivity:

• Use peptide-specific antibodies targeting unique regions of CYP94B1

• Conduct parallel experiments with CYP94B3 and CYP94C1 antibodies to identify differential patterns

• Pre-adsorb antibody with recombinant related proteins to deplete cross-reactive antibodies

• Validate results using genetic knockout lines for each CYP family member

• Perform western blot analysis on samples from plants overexpressing specific CYP family members

• Consider developing monoclonal antibodies with enhanced specificity

What are the best approaches for optimizing CYP94B1 antibody performance in challenging plant tissues?

For challenging plant tissues:

• Modify extraction buffers to account for tissue-specific interfering compounds

• Implement additional purification steps (e.g., ammonium sulfate precipitation, ion exchange)

• Test multiple fixation protocols for immunohistochemistry applications

• Use recombinant protein spike-ins to validate recovery and detection sensitivity

• Consider alternative detection methods (e.g., proximity ligation assay)

• Employ signal amplification technologies for low-abundance situations

• Optimize tissue-specific antigen retrieval methods

How can CYP94B1 antibodies be utilized in studying plant hormone signaling networks?

CYP94B1 antibodies can reveal important aspects of hormone signaling by:

• Tracking CYP94B1 protein accumulation patterns following hormone treatments or stress exposure

• Correlating CYP94B1 protein levels with hormone metabolite profiles across tissues

• Identifying protein interaction partners through co-immunoprecipitation studies

• Analyzing changes in subcellular localization during signaling events

• Quantifying protein abundance in multiple mutant backgrounds to understand genetic interactions

• Monitoring post-translational modifications that may regulate enzyme activity

What methodological approaches enable comparative analysis of CYP94B1 and related enzyme expression in stress responses?

Effective comparative analysis includes:

• Simultaneous protein extraction from control and stressed plant tissues

• Multiplex Western blot analysis with antibodies against CYP94B1, CYP94B3, and CYP94C1

• Quantitative image analysis using appropriate internal loading controls

• Correlation with transcript abundance using RT-qPCR data

• In situ immunolocalization to identify tissue-specific expression patterns

• Metabolomic profiling to correlate enzyme abundance with substrate/product ratios

• Phenotypic characterization of single and multiple cyp94 mutants under stress conditions

How can I develop a biosensor system for monitoring CYP94B1 activity in planta?

To develop an in planta biosensor:

• Utilize CYP94B1 antibodies for immunocapture of the native enzyme

• Combine with fluorogenic or chromogenic substrates that change properties upon hydroxylation

• Adapt piezoimmunosensor approaches used for other CYP family members

• Consider FRET-based systems to detect conformational changes during substrate binding

• Develop genetically encoded fluorescent protein fusions to track CYP94B1 localization

• Implement microfluidic devices for higher throughput analysis

• Validate biosensor data against LC-MS/MS measurements of enzyme products

What experimental design effectively elucidates functional redundancy between CYP94B1 and related enzymes?

An effective experimental design would include:

• Generation and characterization of single, double, and triple mutants (cyp94b1, cyp94b3, cyp94c1)

• Protein expression analysis using specific antibodies against each enzyme

• Detailed metabolite profiling of jasmonate compounds under normal and stress conditions

• Complementation studies with tissue-specific or inducible expression constructs

• In vitro enzyme assays with purified recombinant proteins to compare substrate preferences

• Phenotypic analysis across multiple developmental stages and stress conditions

• Systems biology approaches integrating proteomic, transcriptomic, and metabolomic data

How should experiments be designed to correlate CYP94B1 protein levels with physiological outcomes?

To establish meaningful correlations:

• Implement time-course studies tracking CYP94B1 protein accumulation after stress induction

• Quantify hormone metabolites at the same time points using LC-MS/MS

• Document physiological parameters (growth, defense responses) simultaneously

• Compare wild-type responses with cyp94b1 mutant and overexpression lines

• Use pharmacological approaches with enzyme inhibitors as complementary evidence

• Conduct tissue-specific analyses to identify key sites of CYP94B1 action

• Apply mathematical modeling to establish causal relationships between enzyme levels and outcomes

What controls are essential when using CYP94B1 antibodies in complex experimental systems?

Essential controls include:

• Genetic controls: cyp94b1 knockout plants as negative controls

• Protein controls: purified recombinant CYP94B1 as positive control

• Antibody controls: pre-immune serum, secondary antibody only

• Loading controls: constitutively expressed proteins of similar abundance

• Tissue processing controls: parallel samples processed identically

• Cross-reactivity controls: heterologous expression of related CYP proteins

• Quantification controls: standard curves with known protein concentrations

• Experimental controls: appropriate wild-type comparisons under identical conditions

How might emerging antibody technologies enhance CYP94B1 research?

Emerging technologies with potential impact include:

• Single-domain antibodies (nanobodies) for improved access to conformational epitopes

• Recombinant antibody fragments for enhanced penetration in tissue samples

• Antibody engineering for site-specific conjugation to maintain activity

• Multiparametric imaging using multiplexed antibody panels

• Mass cytometry applications for single-cell protein quantification

• Proximity-dependent labeling for identifying transient interaction partners

• Intrabodies that can track and potentially modulate CYP94B1 activity in living cells

What methodological advances would address current limitations in studying CYP94B1 regulation?

Addressing current limitations requires:

• Development of phospho-specific antibodies targeting regulatory sites

• Improved extraction methods for membrane-associated CYP enzymes

• Single-cell resolution techniques to capture cellular heterogeneity

• Antibody-based chromatin immunoprecipitation to study transcriptional regulation

• Quantitative interactomics approaches to identify regulatory protein partners

• Time-resolved structural studies combining antibody-based purification with cryo-EM

• Development of activity-based protein profiling tools specific for CYP94B1

How can systems biology approaches integrate CYP94B1 antibody data with other molecular datasets?

Integrative approaches should include:

• Correlation of protein abundance data with transcriptomics across conditions

• Integration of proteomic and metabolomic datasets to establish flux control coefficients

• Network analysis incorporating protein-protein interaction data

• Multi-omics data integration to identify regulatory modules

• Machine learning approaches to predict CYP94B1 activity from molecular signatures

• Mathematical modeling to simulate hormone metabolism dynamics

• Comparative analyses across species to identify evolutionarily conserved regulatory mechanisms