CYP82C2 Antibody

What is CYP82C2 and why are antibodies against it valuable in plant research?

CYP82C2 is a cytochrome P450 protein in Arabidopsis thaliana that functions in the biosynthesis of 4-hydroxy indole-3-carbonyl nitrile (4-OH-ICN), a cyanogenic phytoalexin involved in plant defense. CYP82C2 specifically hydroxylates indole-3-carbonyl nitrile (ICN) to generate 4-OH-ICN, a specialized defense metabolite that exhibits antimicrobial properties against bacterial and fungal pathogens .

Antibodies against CYP82C2 are valuable because they enable:

• Detection and localization of CYP82C2 protein in plant tissues

• Monitoring of CYP82C2 expression patterns during pathogen infection

• Investigation of protein-protein interactions in the ICN biosynthetic pathway

• Study of regulatory mechanisms controlling CYP82C2 expression

Methodological approach: When using anti-CYP82C2 antibodies, researchers should consider fixation protocols that preserve membrane protein integrity, as cytochrome P450 proteins are typically membrane-associated. Tissue permeabilization steps should be optimized to ensure antibody access without disrupting protein localization.

What sample preparation techniques are recommended for CYP82C2 antibody applications?

For optimal results with CYP82C2 antibodies, sample preparation should account for the protein's membrane localization and expression patterns:

Methodological note: When working with Arabidopsis samples, consider pathogen treatment with Pseudomonas syringae pv. tomato DC3000 harboring the avrRpm1 avirulence gene (Psta) as this has been shown to significantly upregulate CYP82C2 expression , making it easier to detect the protein in subsequent analyses.

How can researchers validate the specificity of CYP82C2 antibodies?

Validating antibody specificity is crucial given the high sequence similarity within the CYP82C family (CYP82C2, CYP82C3, and CYP82C4 in A. thaliana have >88% protein sequence identity) :

• Western blot comparison: Test the antibody against wild-type plants and cyp82C2 mutant lines to confirm absence of the band in mutants

• Recombinant protein controls: Express and purify recombinant CYP82C2, CYP82C3, and CYP82C4 proteins to test cross-reactivity

• Pre-absorption tests: Pre-incubate the antibody with purified CYP82C2 protein before immunostaining to confirm signal reduction

• Tissue-specific expression analysis: Compare antibody staining patterns with known CYP82C2 expression patterns from transcriptomic data

• Treatment-dependent validation: Verify increased signal in pathogen-treated samples versus reduced signal in iron-deficiency conditions, as CYP82C2 is upregulated by pathogens but not by iron deficiency, unlike CYP82C4

Methodological recommendation: When validating antibody specificity using Western blot, remember that CYP82C2 has an expected molecular weight of approximately 56 kDa based on its 490 amino acid sequence , but observed migration may differ due to post-translational modifications.

How can CYP82C2 antibodies be used to investigate the regulatory neofunctionalization of this enzyme?

CYP82C2 underwent regulatory neofunctionalization following gene duplication from CYP82C4, acquiring pathogen-responsive expression while maintaining similar catalytic capacity . To investigate this evolutionary process:

• Chromatin immunoprecipitation (ChIP) approaches:

  • Perform ChIP with antibodies against histone modifications (H3K4me2, H3K27me3) at the CYP82C2 locus

  • Follow with CYP82C2 antibody immunostaining to correlate protein expression with chromatin state

  • Compare chromatin states between A. thaliana and close relatives lacking 4OH-ICN biosynthesis

• WRKY33 binding analysis:

  • Use CYP82C2 antibodies in combination with WRKY33 antibodies for co-immunoprecipitation

  • Perform sequential ChIP (ChIP-reChIP) to identify regions where both WRKY33 binding and CYP82C2 expression occur

  • Compare these patterns between A. thaliana and A. lyrata, as CYP82C2 is regulated differently in these species

• Transposable element investigation:

  • Use CYP82C2 antibodies to track protein expression in lines with modified EPCOT3 elements

  • Create reporter constructs with and without the EPCOT3 enhancer to study its effects on protein expression

Methodological insight: When investigating regulatory elements like EPCOT3, researchers should combine ChIP approaches using antibodies against both CYP82C2 and transcription factors with DNase I hypersensitivity assays to identify chromatin-accessible regions that may function as enhancers .

What are the best approaches for using CYP82C2 antibodies to study the relationship between pathogen defense and jasmonate signaling?

CYP82C2 functions at the intersection of jasmonate signaling and pathogen defense, as evidenced by the jah1-1 (jasmonic acid-hypersensitive1-1) mutant phenotype . To investigate this relationship:

• Dual immunostaining approaches:

  • Co-immunostain with CYP82C2 antibodies and antibodies against jasmonate signaling components

  • Monitor temporal dynamics of protein expression after pathogen challenge and jasmonate treatment

  • Compare protein localization patterns in different cell types and tissues

• Protein complex analysis:

  • Use CYP82C2 antibodies for co-immunoprecipitation followed by mass spectrometry

  • Identify interaction partners under different conditions (pathogen infection vs. jasmonate treatment)

  • Validate interactions with yeast two-hybrid or bimolecular fluorescence complementation

• Metabolite correlation studies:

  • Combine CYP82C2 protein quantification via immunoblotting with metabolic profiling

  • Track correlations between protein levels and accumulation of 4OH-ICN, indole glucosinolates, and jasmonate metabolites

  • Compare these profiles between wild-type, jah1-1 mutants, and CYP82C2-overexpressing plants

Methodological recommendation: When studying jasmonate responses, standardize treatments using 50-100 μM methyl jasmonate and collect tissues at multiple time points (1, 3, 6, 12, and 24 hours) to capture the dynamic nature of the response .

How can epitope mapping improve CYP82C2 antibody design for distinguishing between closely related cytochrome P450 family members?

Distinguishing CYP82C2 from its close paralogs (CYP82C3 and CYP82C4) requires careful epitope selection and antibody validation:

• Computational epitope mapping approach:

  • Align sequences of CYP82C2, CYP82C3, and CYP82C4 to identify unique regions

  • Use epitope prediction algorithms (similar to ASEP, BEPAR, ABEpar) to identify CYP82C2-specific surface-exposed epitopes

  • Design peptide antigens based on these unique regions for antibody production

• Advanced validation methods:

  • Perform competitive ELISA using peptides from homologous regions of CYP82C3 and CYP82C4

  • Use synthetic peptide arrays to fine-map the exact epitope recognized by the antibody

  • Test antibody binding against recombinant proteins with site-directed mutations in the predicted epitope region

• CDR walking for antibody optimization:

  • Apply CDR walking techniques to systematically mutate complementarity-determining regions (CDRs) in monoclonal antibodies

  • Screen mutants for improved specificity against CYP82C2 versus CYP82C3 and CYP82C4

  • Use the optimized antibody for high-resolution studies of CYP82C2 localization and dynamics

Methodological insight: When designing peptide antigens for CYP82C2-specific antibodies, focus on regions outside the catalytic domain that show the greatest sequence divergence from CYP82C3 and CYP82C4. The N-terminal region (residues 1-50) and the loop regions between conserved helices typically offer the best targets for specific antibody generation.

Why might CYP82C2 antibodies show inconsistent results in different experimental systems?

Several factors can contribute to inconsistent results when using CYP82C2 antibodies:

• Protein expression variability:

  • CYP82C2 expression is highly pathogen-inducible and may be low in untreated samples

  • Expression patterns differ significantly between A. thaliana and related species

  • WRKY33-dependent regulation means expression varies with pathogen exposure timing and intensity

• Technical considerations:

  • Membrane protein extraction efficiency affects detection sensitivity

  • Sample preparation methods may denature epitopes recognized by the antibody

  • Buffer conditions (pH, salt concentration, detergent type) can affect antibody binding

• Biological factors:

  • Post-translational modifications may mask epitopes

  • Protein-protein interactions may block antibody access to the epitope

  • Rapid turnover of the protein after pathogen induction

Methodological solutions:

• Include positive controls (pathogen-treated samples) where CYP82C2 expression is known to be high

• Optimize protein extraction with different detergent combinations

• Test different antibody dilutions and incubation conditions

• Consider using epitope-tagged CYP82C2 constructs for comparative analysis

What strategies can address cross-reactivity issues with CYP82C2 antibodies?

Cross-reactivity with other cytochrome P450 family members is a common challenge:

• Antibody purification approaches:

  • Perform affinity purification using immobilized CYP82C2-specific peptides

  • Deplete cross-reactive antibodies through adsorption with recombinant CYP82C3 and CYP82C4

  • Use sequential affinity purification to isolate highly specific antibody fractions

• Experimental design modifications:

  • Include proper negative controls (cyp82C2 mutant lines)

  • Use differential expression conditions (pathogen treatment vs. iron deficiency) to distinguish between CYP82C2 and CYP82C4

  • Combine antibody-based detection with targeted proteomics for validation

• Alternative approaches when cross-reactivity persists:

  • Develop epitope-tagged CYP82C2 constructs under native promoters

  • Use CRISPR/Cas9 to tag the endogenous CYP82C2 gene with a specific epitope

  • Employ RNA-protein co-detection methods to simultaneously visualize the transcript and protein

Methodological insight: When persistent cross-reactivity occurs, competitive Western blotting can help assess the degree of cross-reactivity. Pre-incubate the antibody with increasing concentrations of purified competitor proteins (CYP82C3 or CYP82C4) before probing for CYP82C2. The concentration of competitor protein needed to reduce signal provides a quantitative measure of cross-reactivity.

How can CYP82C2 antibodies be used to investigate the evolutionary history of specialized metabolic pathways?

CYP82C2 represents a case of gene duplication and neofunctionalization contributing to species-specific metabolic diversity . To investigate evolutionary aspects:

• Comparative immunohistochemistry approaches:

  • Use CYP82C2 antibodies on tissue sections from multiple Brassicaceae species

  • Compare protein expression patterns with metabolite profiles across species

  • Correlate CYP82C2 detection with the presence of 4OH-ICN production

• Protein conservation analysis:

  • Test antibody cross-reactivity with homologs from diverse species

  • Correlate antibody binding with sequence conservation in epitope regions

  • Use phylogenetic analysis to map antibody reactivity to evolutionary distances

• Structure-function studies:

  • Use antibodies to immunoprecipitate CYP82C2 from different species for activity assays

  • Compare substrate specificity of immunopurified enzymes

  • Correlate catalytic differences with structural variations

Species distribution of 4OH-ICN biosynthesis and CYP82C2 homologs:

Methodological recommendation: When studying protein evolution, combine antibody-based detection with activity assays to distinguish between sequence conservation and functional conservation. Proteins may be recognized by antibodies but have evolved different substrate specificities or regulatory mechanisms.

How can CYP82C2 antibodies contribute to understanding the interplay between transposable elements and regulatory network evolution?

The EPCOT3 transposable element functions as an enhancer for CYP82C2 expression in A. thaliana, demonstrating how TEs can rewire regulatory networks . To investigate this phenomenon:

• Chromatin structure analysis:

  • Combine CYP82C2 antibody-based ChIP with assays for chromatin accessibility (ATAC-seq, DNase-seq)

  • Map the correlation between chromatin state at the EPCOT3 locus and CYP82C2 protein levels

  • Compare chromatin landscapes between species with and without the EPCOT3 insertion

• Transcription factor binding studies:

  • Use WRKY33 and CYP82C2 antibodies for sequential ChIP experiments

  • Identify co-occupied genomic regions that may indicate direct regulation

  • Compare binding patterns between wild-type and mutant lines with modified EPCOT3 elements

• Epigenetic regulation analysis:

  • Track histone modifications (H3K4me2, H3K27me3) at the EPCOT3 locus during pathogen response

  • Correlate these modifications with CYP82C2 protein levels detected by immunoblotting

  • Investigate the effect of epigenetic inhibitors on CYP82C2 expression and protein accumulation

Methodological insight: To directly investigate the role of EPCOT3, researchers can use CRISPR/Cas9 to delete or modify this element in A. thaliana and then use CYP82C2 antibodies to assess the impact on protein expression in response to pathogens. Complementary approaches might include introducing the EPCOT3 element into A. lyrata to test if it can confer pathogen-responsive CYP82C2 expression.

What are the most effective protocols for using CYP82C2 antibodies in multi-omics integration studies?

Integrating antibody-based protein detection with other omics approaches provides comprehensive insights into CYP82C2 function:

• Protein-metabolite correlation studies:

  • Quantify CYP82C2 protein levels using calibrated immunoblotting

  • Perform parallel metabolomics to measure ICN, 4OH-ICN, and related compounds

  • Calculate correlation coefficients between protein abundance and metabolite levels across conditions

• Spatial multi-omics approaches:

  • Use immunohistochemistry to map CYP82C2 protein localization

  • Perform laser capture microdissection of immunopositive regions

  • Analyze transcriptomes and metabolomes from these specific cellular populations

• Temporal dynamics integration:

  • Collect time-course samples after pathogen treatment

  • Process parallel samples for CYP82C2 protein detection, transcript quantification, and metabolite analysis

  • Develop mathematical models of the relationship between transcript, protein, and metabolite dynamics

Integration framework for CYP82C2 studies:

Methodological recommendation: When designing multi-omics studies, ensure that sample collection methods are compatible with all planned analyses. For example, fixation methods for immunohistochemistry may interfere with metabolite extraction, necessitating parallel sample processing streams from the same experimental cohort.