CYP71 Antibody

What is the CYP71 family and why is it significant in plant research?

The CYP71 family represents the largest cytochrome P450 clade in plants, with diverse roles in specialized metabolism. Based on phylogenetic analysis, CYP71 proteins are organized into multiple subclades that have evolved for specific metabolic functions. For example, in Cichorium intybus (chicory), the CYP71 clade contains 26 genes divided into distinct functional groups including GAO (CYP71AV subclade), COS (CYP71BL subclade), and KLS (CYP71BZ subclade) .

These enzymes catalyze oxidation reactions in various specialized metabolic pathways, making them critical targets for understanding plant biochemistry, stress responses, and metabolic engineering. Research interest in CYP71 proteins has intensified due to their roles in synthesizing bioactive compounds with pharmaceutical and agricultural applications.

What are the main detection methods for CYP71 proteins in plant tissues?

CYP71 proteins can be detected through several complementary approaches:

• Immunological techniques: Western blotting, immunohistochemistry, and ELISA using specific antibodies against CYP71 proteins

• Mass spectrometry: For precise identification and quantification of specific CYP71 isoforms

• Activity-based assays: Using specific substrates to measure enzymatic activity

• Gene expression analysis: RT-qPCR or RNA-seq to measure transcript levels as a proxy for protein expression

When selecting detection methods, researchers should consider that different CYP71 subfamilies may require specific protocols. For example, when studying MeJA-inducible CYP71 proteins, time-course experiments revealed maximum induction between 6-24 hours post-treatment in chicory seedlings .

How do I determine the specificity of a commercial CYP71 antibody?

Determining antibody specificity is crucial for reliable results, especially with the high sequence similarity among CYP71 family members:

• Sequence alignment analysis: Compare the immunogen sequence with other CYP71 family members to predict potential cross-reactivity

• Knockout/knockdown validation: Test the antibody in plant tissues with CRISPR-edited or silenced CYP71 genes

• Heterologous expression: Express recombinant CYP71 proteins in systems like N. benthamiana and use as positive controls

• Cross-reactivity testing: Test against closely related CYP71 subfamily members

For example, in chicory research, expression in N. benthamiana was used to validate CYP71 function, providing material for subsequent antibody validation . This approach can distinguish between closely related paralogs, such as the CYP71BL subclade members (CYP71BL11, CYP71BL10, CYP71BL3, and CYP71BL12) .

How can CYP71 antibodies help characterize specialized metabolic pathways?

CYP71 antibodies serve as powerful tools for elucidating specialized metabolic pathways through multiple approaches:

• Protein localization: Immunohistochemistry can reveal tissue-specific and subcellular localization patterns

• Protein-protein interaction studies: Co-immunoprecipitation followed by mass spectrometry can identify metabolic complexes

• Enzyme regulation: Western blot analysis of CYP71 proteins under different conditions can reveal post-translational modifications

• Metabolic flux analysis: Combining antibody-based protein quantification with metabolite profiling

Research on chicory has demonstrated that multiple CYP71 paralogs (CYP71BZ subclade) with high sequence similarity can have distinct functions in sesquiterpene lactone biosynthesis . Using specific antibodies allows researchers to track the expression and localization of these paralogs under different conditions, providing insights into their physiological roles.

What are the optimal sample preparation techniques for CYP71 antibody applications?

CYP71 proteins are membrane-associated and often present at low abundance, requiring specific sample preparation protocols:

For Western blotting:

• Use microsomal preparation techniques to enrich membrane proteins

• Include protease inhibitors to prevent degradation

• Avoid boiling samples (heat at 37°C instead) to prevent aggregation

• Use 5-10% SDS-PAGE gels for optimal separation

• Transfer proteins at low voltage (30V) overnight for efficient transfer

For immunohistochemistry:

• Use paraformaldehyde fixation at 4% concentration

• Consider antigen retrieval with TE buffer pH 9.0 (similar to protocols for other P450 enzymes)

• Block with 5% BSA in PBS to reduce background

• Incubate primary antibody at 4°C overnight

• Use tyramide signal amplification for low-abundance proteins

These protocols have been adapted from successful approaches with other cytochrome P450 antibodies and should be optimized for specific CYP71 subfamilies.

How can I develop antibodies against specific CYP71 subfamily members?

Developing specific antibodies against CYP71 subfamily members requires careful design:

• Immunogen selection: Target unique regions (typically N-terminal or C-terminal) that differ between paralogs

• Multiple immunogen approach: Generate antibodies against 2-3 different regions to increase specificity

• Recombinant protein expression: Express full-length CDS of target CYP71 in heterologous systems like E. coli or N. benthamiana

• Affinity purification: Use recombinant proteins for affinity purification of antibodies

The following table shows recommended regions for immunogen design based on sequence analysis of CYP71 subfamilies:

Success has been demonstrated with the heterologous expression approach, as shown in chicory research where full-length CDS was amplified from cDNA and expressed in N. benthamiana for functional validation .

How do I address cross-reactivity issues with CYP71 antibodies?

Cross-reactivity is common with CYP71 antibodies due to high sequence similarity between subfamily members:

• Pre-absorption: Incubate antibody with recombinant proteins from closely related CYP71 members

• Epitope mapping: Identify the specific epitope recognized by the antibody to predict cross-reactivity

• Peptide competition: Use synthetic peptides corresponding to the immunogen to confirm specificity

• Dual validation: Combine antibody detection with RNA expression data for comprehensive analysis

For example, in the CYP71BZ subclade, ten potential paralogs were identified in C. intybus, divided into three subclades (I to III) . These paralogs show various degrees of sequence similarity, requiring careful antibody validation to distinguish between them.

What are the optimal dilutions and incubation conditions for CYP71 antibody applications?

Based on protocols developed for other cytochrome P450 antibodies, the following starting conditions are recommended:

Western Blot:

• Primary antibody dilution: 1:500-1:1500

• Incubation: Overnight at 4°C

• Secondary antibody dilution: 1:5000-1:10000

• Detection: Enhanced chemiluminescence

Immunohistochemistry:

• Primary antibody dilution: 1:20-1:200

• Incubation: 24-48 hours at 4°C

• Secondary antibody dilution: 1:500

• Detection: DAB or fluorescent secondary antibodies

Immunoprecipitation:

• Antibody amount: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Incubation: 4 hours to overnight at 4°C

• Protein A/G beads: 20-50 μl of slurry

These recommendations are based on protocols for other P450 antibodies and should be optimized for each specific CYP71 antibody and sample type.

How do I interpret contradictory results between antibody detection and gene expression data?

Discrepancies between antibody detection and gene expression data for CYP71 proteins can occur for several reasons:

• Post-transcriptional regulation: Check for microRNA targeting CYP71 transcripts

• Protein stability differences: Assess protein half-life through cycloheximide chase experiments

• Translational efficiency: Analyze polysome association of CYP71 mRNAs

• Technical limitations: Antibody sensitivity or specificity issues may cause false negatives

Recent research in chicory shows that MeJA treatment affected gene expression of CYP71 family members differently across plant varieties, with some genes showing rapid induction and others displaying delayed responses . This demonstrates the complexity of CYP71 regulation and the need for multiple analytical approaches.

How can CYP71 antibodies be used to study protein-protein interactions in specialized metabolic pathways?

CYP71 proteins often function within metabolic complexes or metabolons. To study these interactions:

• Co-immunoprecipitation: Use CYP71 antibodies to pull down protein complexes

• Proximity labeling: Combine with BioID or APEX2 approaches to identify proximal proteins

• In situ PLA (Proximity Ligation Assay): Visualize interactions between CYP71 and candidate partners

• Blue native PAGE: Analyze intact protein complexes containing CYP71 proteins

Research on CYP71BZ proteins (KLS) in chicory has identified three functional genes (CYP71BZ25, CYP71BZ26, and CYP71BZ27) that are tandemly duplicated and located on chromosome 5 . These proteins likely interact with other enzymes in sesquiterpene lactone biosynthesis, making them ideal candidates for protein-protein interaction studies.

What are the considerations for using CYP71 antibodies across different plant species?

CYP71 proteins show varying degrees of conservation across plant species, affecting antibody cross-reactivity:

• Sequence alignment: Compare the immunogen sequence across target species

• Western blot validation: Test the antibody on samples from each target species

• Epitope conservation analysis: Predict antibody binding based on epitope conservation

• Recombinant protein controls: Express the orthologous proteins from each species as positive controls

For example, studies comparing CYP71 genes between Cichorium intybus and Lactuca sativa revealed varying degrees of conservation - some subclades showed high orthology while others had species-specific expansions . The CYP71BL subclade showed three lettuce orthologs that were all MeJA-inducible, potentially allowing cross-species antibody applications .

How can CYP71 antibodies be used to understand regulation of specialized metabolism under stress conditions?

CYP71 antibodies provide valuable tools for studying stress responses:

• Time-course analysis: Track CYP71 protein levels at different time points after stress treatment

• Tissue-specific changes: Use immunohistochemistry to identify tissue-specific regulation

• Post-translational modifications: Detect phosphorylation or other modifications using specific antibodies

• Subcellular relocalization: Examine potential changes in CYP71 localization under stress

Research in chicory demonstrated that multiple CYP71 genes are induced by methyl jasmonate (MeJA), a stress-related hormone . For instance, CYP71BL10 and CYP71BL3 were MeJA-inducible in both industrial chicory and witloof, while CYP81BZ19 and CYP71BZ18 showed similar induction patterns . Antibodies against these proteins would allow researchers to determine if protein levels mirror the transcriptional changes observed.

How can CYP71 antibodies contribute to metabolic engineering efforts?

CYP71 antibodies can support metabolic engineering in several ways:

• Protein expression optimization: Monitor protein levels during optimization of expression constructs

• Subcellular targeting validation: Confirm correct localization of engineered CYP71 proteins

• Protein stability assessment: Evaluate the stability of modified CYP71 proteins

• Metabolic burden analysis: Correlate CYP71 protein levels with pathway performance and cellular stress

The detailed characterization of CYP71 family members in chicory, including functional testing through heterologous expression in N. benthamiana, provides a foundation for such engineering approaches . For example, the identification of functional CYP71BZ enzymes (KLS) enables their targeted expression in heterologous systems for sesquiterpene lactone production.

What are the prospects for developing autoantibody assays related to CYP71 exposure in occupational settings?

While not directly documented for CYP71, research on other cytochrome P450 enzymes suggests potential approaches:

• Epitope mapping: Identify potentially immunogenic regions in CYP71 proteins

• ELISA development: Design assays to detect human antibodies against CYP71 proteins

• Cross-reactivity analysis: Assess potential cross-reactivity with human P450 enzymes

• Exposure biomarker validation: Correlate antibody levels with known exposure metrics

Studies on trichloroethylene exposure have demonstrated that anti-CYP2E1 autoantibodies can serve as biomarkers of exposure, with levels significantly higher in exposed individuals compared to non-exposed controls . Similar approaches could potentially be developed for plant CYP71 exposure scenarios, particularly in agricultural or industrial settings where workers may be exposed to plant materials containing CYP71 proteins.