CYP86A7 Antibody

What is CYP86A7 and what is its functional role in plants?

CYP86A7 (At1g63710) is a member of the CYP86A subfamily of cytochrome P450 enzymes in Arabidopsis thaliana. It functions as a fatty acid ω-hydroxylase involved in cutin biosynthesis . The enzyme catalyzes the hydroxylation of fatty acids, which is a critical step in the formation of cutin polymers. These polymers contribute to the plant cuticle, which plays essential roles in:

• Protection against water loss

• Defense against pathogen invasion

• Regulation of organ development

• Plant-environment interactions

The protein belongs to non-A type P450s with significant sequence homology to fatty acid- and alkane-metabolizing CYP4 proteins in mammals and CYP52 proteins in fungi . Functionally, CYP86A7 is closely related to other characterized fatty acid hydroxylases in plants that participate in cuticle formation and stress responses.

How is CYP86A7 structurally related to other members of the CYP86A subfamily?

CYP86A7 shares varying degrees of sequence identity with other members of the Arabidopsis CYP86A subfamily:

CYP86A7 consists of 524 amino acids, which is 11 residues longer than CYP86A1 (513 aa) but shorter than CYP86A8 (537 aa), CYP86A2 (553 aa), and CYP86A4 (557 aa) . The length differences primarily occur at the C-terminus. Analysis of substrate recognition sites (SRS) shows that CYP86A7 is most variable in SRS2, SRS3, and SRS6 compared to other subfamily members, suggesting potential differences in substrate specificity .

Unlike CYP86A1, CYP86A2, and CYP86A4, which contain introns, the CYP86A7 gene has no introns, a feature it shares only with CYP86A8 among subfamily members .

How is CYP86A7 expression regulated in plants?

CYP86A7 expression is regulated by several factors:

What experimental approaches can be used to study CYP86A7 expression patterns?

Several methods have been employed to study CYP86A7 expression:

• RT-PCR and qRT-PCR: Semi-quantitative and quantitative PCR methods allow for the measurement of CYP86A7 transcript levels in different tissues or under various conditions .

• Microarray analysis: This approach has been used to identify CYP86A7 as a target of transcription factors like WIN1/SHN1 .

• Promoter-reporter constructs: Fusing the CYP86A7 promoter to reporter genes like GFP or luciferase can help visualize expression patterns in planta .

• In situ hybridization: This technique allows for the visualization of mRNA expression in tissue sections, providing spatial information on gene expression .

• Chromatin immunoprecipitation (ChIP): Can be used to identify transcription factors that bind to the CYP86A7 promoter .

What are the most effective approaches for generating antibodies against CYP86A7?

Based on comprehensive antibody production experiences for Arabidopsis proteins:

• Recombinant protein approach (recommended):

  • Identify potential antigenic regions using bioinformatic tools (like DNASTAR)

  • Confirm uniqueness by BLAST analysis (aim for <40% similarity to other proteins)

  • Clone and express the selected region as a 6xHis-tagged recombinant protein

  • Affinity purify the resulting antibodies against the purified recombinant protein

This approach has shown a 55% success rate for Arabidopsis protein antibodies, compared to the much lower success of peptide-based approaches .

• Important considerations:

  • Select sequences of approximately 100 amino acids when possible

  • For membrane proteins like CYP86A7, avoid transmembrane domains

  • Consider protein secondary structure when selecting antigenic regions

  • Affinity purification is crucial for obtaining specific antibodies

The peptide approach (using 12-15 amino acid synthetic peptides) showed very poor success rates in plant protein antibody production, with only 1 out of 24 antibodies working satisfactorily in one study .

How can I validate the specificity of a CYP86A7 antibody?

Multiple validation methods should be employed:

• Dot blot analysis: Test antibody detection limits using purified recombinant protein at various dilutions (picogram to nanogram range) .

• Western blotting validation:

  • Use wild-type Arabidopsis tissue extracts to confirm detection of a band at the expected size (~58 kDa for CYP86A7)

  • Include cyp86a7 knockout mutant samples as negative controls

  • Test antibody cross-reactivity with other CYP86A subfamily members expressed as recombinant proteins

• Immunolocalization validation:

  • Compare signal patterns in wild-type and cyp86a7 mutant tissues

  • Use appropriate fixation methods (paraformaldehyde for membrane proteins)

  • Include pre-immune serum controls and peptide competition assays

• ChIP validation: If using the antibody for chromatin immunoprecipitation, validate by:

  • Confirming enrichment of known CYP86A7-associated DNA fragments

  • Including no-antibody and IgG controls

  • Validating with tagged CYP86A7 constructs in transgenic plants

How can I optimize Western blot protocols for CYP86A7 detection?

Optimizing Western blot detection of CYP86A7 requires specific considerations:

• Sample preparation:

  • Use freshly prepared tissue extracts with protease inhibitors

  • For membrane proteins like CYP86A7, include appropriate detergents (0.1-1% Triton X-100 or NP-40)

  • Consider enriching membrane fractions by ultracentrifugation

  • Heat samples at 37°C instead of boiling to prevent aggregation of membrane proteins

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal resolution

  • Include molecular weight markers spanning 40-70 kDa range

  • Consider gradient gels for better separation

• Transfer and detection:

  • Optimize transfer conditions for membrane proteins (longer transfer times or semi-dry transfer)

  • Use PVDF membranes instead of nitrocellulose for better protein retention

  • Block with 5% non-fat milk or BSA in TBS-T

  • Determine optimal antibody dilution (typically starting at 1:1000 for affinity-purified antibodies)

  • Include appropriate positive controls (recombinant CYP86A7) and negative controls (cyp86a7 mutant extract)

What are effective immunolocalization protocols for studying CYP86A7 distribution in plant tissues?

For successful immunolocalization of CYP86A7:

• Tissue fixation and embedding:

  • Fix tissues in 4% paraformaldehyde in PBS for 2-4 hours

  • For membrane proteins like CYP86A7, avoid strong fixatives that may mask epitopes

  • Embed in paraffin or prepare cryosections depending on tissue type

  • For root tissues, whole-mount immunolocalization may be effective

• Antigen retrieval and blocking:

  • Perform antigen retrieval using citrate buffer (pH 6.0) if needed

  • Block with 3% BSA or normal serum in PBS with 0.1% Triton X-100

  • Include steps to block endogenous peroxidase if using HRP-based detection

• Antibody incubation and detection:

  • Incubate with primary antibody at optimized dilution (typically 1:50-1:200) overnight at 4°C

  • Use fluorescently-labeled secondary antibodies for better resolution

  • Include DAPI or other nuclear stains for reference

  • Examine multiple cell types and developmental stages

  • Always compare with negative controls (pre-immune serum, cyp86a7 mutant)

How can ChIP-seq be performed using CYP86A7 antibodies to study protein-DNA interactions?

While CYP86A7 itself is not a transcription factor, this question addresses experimental design for researchers interested in studying potential chromatin associations of CYP86A7 or related proteins:

• Experimental design considerations:

  • Cross-link plant tissue with 1% formaldehyde to preserve protein-DNA interactions

  • Isolate and fragment chromatin (200-500 bp fragments)

  • Immunoprecipitate using optimized amounts of CYP86A7 antibody

  • Include appropriate controls (input DNA, IgG control, negative genomic regions)

• Validation of ChIP efficiency:

  • Perform qPCR on known regions before proceeding to sequencing

  • Include positive controls such as promoters of genes regulated by transcription factors known to interact with CYP86A7

  • Verify enrichment compared to background

• Sequencing and data analysis:

  • Prepare ChIP-seq libraries following standard protocols

  • Perform paired-end sequencing for better alignment

  • Analyze data using specialized software (MACS2, Homer)

  • Validate peaks with motif analysis and comparison to known binding sites

• Biological validation:

  • Confirm selected binding sites using reporter gene assays

  • Perform functional analysis of identified target genes

  • Consider protein complex analysis to identify co-factors

How can I investigate the role of CYP86A7 in plant stress responses using antibody-based approaches?

To study CYP86A7's involvement in stress responses:

• Protein expression profiling:

  • Expose plants to various stresses (drought, rehydration, pathogen exposure, etc.)

  • Collect tissue samples at multiple time points

  • Perform Western blot analysis to quantify CYP86A7 protein levels

  • Compare protein expression with transcript levels to identify post-transcriptional regulation

• Subcellular localization changes:

  • Use immunolocalization to track potential relocalization of CYP86A7 during stress

  • Compare with the localization pattern of other CYP86A subfamily members

  • Consider co-localization with stress-related markers

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation using CYP86A7 antibodies

  • Identify stress-specific interaction partners by mass spectrometry

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

• Post-translational modifications:

  • Use immunoprecipitation followed by mass spectrometry to identify PTMs

  • Develop phospho-specific or other modification-specific antibodies if relevant

  • Compare PTM patterns under normal and stress conditions

What approaches can be used to study the potential role of CYP86A7 in transcriptional regulation?

Although CYP86A7 is an enzyme rather than a transcription factor, understanding its relationship with transcriptional networks is valuable:

• ChIP analysis of transcription factors binding CYP86A7 promoter:

  • Identify transcription factors predicted to bind the CYP86A7 promoter (like WIN1/SHN1)

  • Perform ChIP using antibodies against these transcription factors

  • Test enrichment of CYP86A7 promoter fragments

• Promoter analysis:

  • Generate deletion series of the CYP86A7 promoter fused to reporter genes

  • Identify minimal promoter elements required for expression

  • Perform site-directed mutagenesis of predicted binding sites

• Transcriptome analysis in cyp86a7 mutants:

  • Compare gene expression profiles between wild-type and cyp86a7 mutant plants

  • Focus analysis on cutin biosynthesis pathways and stress response genes

  • Validate key differentially expressed genes by qRT-PCR

• Integration with metabolite profiles:

  • Correlate CYP86A7 protein levels with changes in cutin monomers

  • Investigate feedback regulation mechanisms where cutin components might influence CYP86A7 expression

  • Develop experimental systems to test such regulatory feedback loops

What are common challenges in detecting CYP86A7 by Western blotting and how can they be addressed?

Researchers often encounter specific challenges when working with cytochrome P450 proteins like CYP86A7:

• Multiple or unexpected bands:

  • Cause: Protein degradation, cross-reactivity, post-translational modifications

  • Solution: Use fresh tissue extracts with protease inhibitors, optimize antibody concentration, perform peptide competition assays

• No signal detection:

  • Cause: Low protein abundance, epitope masking, inefficient transfer

  • Solution: Enrich membrane fractions, test different extraction buffers, optimize transfer conditions, increase antibody concentration

• High background:

  • Cause: Non-specific binding, insufficient blocking, contaminated antibody

  • Solution: Increase blocking time/concentration, use alternative blocking agents, purify antibody, optimize washing steps

• Inconsistent results between experiments:

  • Cause: Variation in protein extraction efficiency, environmental effects on gene expression

  • Solution: Standardize growth conditions, use internal loading controls, pool samples from multiple plants

How can I resolve cross-reactivity issues with antibodies against CYP86A family members?

Due to the high sequence similarity between CYP86A members, cross-reactivity can be a significant challenge:

• Epitope selection strategies:

  • Target regions with lowest sequence conservation between family members

  • Avoid highly conserved functional domains when possible

  • Consider raising antibodies against C-terminal regions, which show greater variability

• Antibody purification approaches:

  • Perform sequential affinity purification:

    • First purify against the immunogen

    • Then perform negative selection against recombinant proteins of other family members

  • Cross-adsorption with extracts from plants overexpressing other CYP86A proteins

• Validation in genetic backgrounds:

  • Test antibodies in cyp86a7 knockout mutants (should show no signal)

  • Test in plants overexpressing CYP86A7 (should show enhanced signal)

  • Compare with plants lacking or overexpressing other CYP86A family members

• Computational prediction:

  • Use epitope prediction software to identify CYP86A7-specific regions

  • Perform detailed sequence alignments focusing on surface-exposed regions

  • Consider structural models to identify accessible, unique epitopes

How might new antibody technologies advance our understanding of CYP86A7 function?

Emerging technologies offer new possibilities for studying CYP86A7:

• Nanobodies and single-domain antibodies:

  • Smaller size allows better tissue penetration for in situ studies

  • Can recognize epitopes inaccessible to conventional antibodies

  • Potential for direct fusion to fluorescent proteins for live imaging

• Proximity labeling approaches:

  • Antibody-mediated targeting of enzymes like BioID or APEX2

  • Allows identification of proteins in close proximity to CYP86A7

  • Can reveal membrane-associated complexes and transient interactions

• Single-molecule tracking:

  • Combine high-affinity antibody fragments with quantum dots

  • Track CYP86A7 movement within cellular membranes

  • Study dynamics of protein localization during stress responses

• Intrabodies and targeted protein degradation:

  • Express antibody fragments intracellularly to modulate CYP86A7 function

  • Use antibody-based targeted protein degradation systems as alternatives to genetic knockouts

  • Study acute loss of CYP86A7 function in specific tissues or developmental stages

What are promising approaches for studying post-translational modifications of CYP86A7?

Investigating PTMs of CYP86A7 requires specialized techniques:

• Mass spectrometry approaches:

  • Immunoprecipitate CYP86A7 using validated antibodies

  • Analyze by LC-MS/MS to identify phosphorylation, ubiquitination, or other modifications

  • Compare PTM profiles under different stress conditions or developmental stages

• Modification-specific antibodies:

  • Develop antibodies against predicted phosphorylation sites

  • Use these to monitor changes in CYP86A7 phosphorylation status

  • Apply in Western blotting and immunolocalization studies

• In vitro modification assays:

  • Express recombinant CYP86A7 and test as substrate for known kinases

  • Identify enzymes responsible for specific modifications

  • Validate findings in planta using genetic approaches

• Functional consequences of PTMs:

  • Create phosphomimetic or phospho-null mutations at identified sites

  • Express modified versions in cyp86a7 mutants

  • Analyze effects on protein activity, stability, and localization