CYP86A1 Antibody
Product Specs
Target Background
CYP86A1 (At5g58860) has been identified as a crucial enzyme in the biosynthesis of aliphatic root suberin in Arabidopsis thaliana. Further details can be found in the following publication:
Q&A
Basic Research Questions
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What is CYP86A1 and what functional role does it play in plants?
CYP86A1 is a cytochrome P450 monooxygenase that functions as a fatty acid ω-hydroxylase primarily involved in suberin biosynthesis. It catalyzes the ω-hydroxylation of saturated and unsaturated fatty acids with chain lengths ranging from C12 to C18, a critical step in creating the monomers required for suberin formation . In Arabidopsis, CYP86A1 (also known as HORST - hydroxylase of root suberized tissue) is expressed predominantly in the root endodermis where it contributes to the formation of apoplastic barriers . Functionally, CYP86A1 participates in three sequential oxidation reactions that add hydroxyl groups to fatty acids, thereby enabling their incorporation into suberin polymers that form protective barriers in plant roots .
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What are the recommended validation techniques for CYP86A1 antibodies?
When validating CYP86A1 antibodies, researchers should employ multiple complementary approaches:
| Validation Method | Procedure | Expected Result | Limitations |
|---|---|---|---|
| Western Blot with knockout controls | Compare wild-type vs. cyp86a1 mutant samples | Signal present in WT, absent in mutant | May not validate for non-denaturing applications |
| Immunohistochemistry (IHC) | Tissue-specific localization | Signal in endodermal cells of roots | Requires proper fixation optimization |
| CRISPR-Cas9 validation | Generate knockout cell lines and test antibody | Reduction/elimination of signal in knockout lines | Time-consuming to generate proper controls |
| Independent antibody approach | Compare staining patterns with antibodies targeting different epitopes | Similar localization patterns | Requires availability of multiple validated antibodies |
The most rigorous approach combines genetic strategies with independent antibody validation. Current quality standards increasingly require demonstrating specificity through genetic knockouts (CRISPR-Cas9, RNAi) alongside traditional methods . Additionally, recombinant antibody technology offers improved reproducibility compared to traditional hybridoma-derived antibodies .
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What are the optimal working dilutions for CYP86A1 antibodies in common applications?
Based on validated protocols, the following dilution ranges are recommended:
| Application | Recommended Dilution | Sample Types | Notes |
|---|---|---|---|
| Western Blot | 1:1000-1:5000 | Plant tissue extracts | Higher concentrations may be needed for low abundance samples |
| Immunohistochemistry | 1:20-1:200 | Fixed plant tissues | Optimization required for each tissue type |
| Immunofluorescence | 1:100 | Cellular preparations | Best results with fresh samples |
| ELISA | Application-specific | Protein extracts | Varies by conjugated antibody type (HRP: higher dilution; Biotin: lower) |
It's essential to perform a dilution series to determine optimal concentration for each specific experimental setup and plant species . For cross-species applications, validation at multiple dilutions is recommended due to potential affinity differences.
Advanced Research Questions
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How can researchers effectively study transcription factor regulation of CYP86A1 expression?
Multiple approaches have proven effective for examining transcription factor regulation of CYP86A1:
Research has identified several transcription factors that regulate CYP86A1 expression, including MYB41, MYB107, MYC2, and WRKY33. Promoter analysis typically reveals MYB recognition elements (solid lines) and MYC recognition elements (boxed regions) that serve as binding sites . For meaningful results, combine binding assays (Y1H, ChIP) with functional validation through reporter assays and gene expression analysis in TF mutant backgrounds .
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What methodological approaches can confirm CYP86A1's role in suberin biosynthesis?
Establishing CYP86A1's role in suberin biosynthesis requires a multi-faceted experimental approach:
The most convincing demonstrations combine genetic knockout approaches with chemical analysis of suberin monomers and functional complementation. Advanced approaches might include the use of barrier function assays, such as fluorescein diacetate (FDA) uptake, which has demonstrated that only ~10% of endodermal cells in wild-type plants allow FDA penetration compared to significantly higher percentages in cyp86a1 mutants .
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How should researchers interpret CYP86A1 antibody results in stress response studies?
When studying CYP86A1 in stress response contexts, careful experimental design and interpretation are essential:
For robust interpretation, use both transcript and protein level analysis, as post-transcriptional regulation may occur. In studies with GbCYP86A1-1 from cotton, researchers observed that pathogen exposure (Verticillium dahliae) induced expression specifically in roots . Similarly, salt stress studies have shown that CYP94B1 (a related cytochrome P450) and CYP86A1 expression significantly increases in endodermal cells after treatment . Always correlate antibody-based protein detection with functional measurements of suberin content and barrier properties.
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What are critical considerations when using CYP86A1 antibodies for co-localization studies?
Co-localization studies with CYP86A1 require careful experimental design:
Recent studies have found that CYP86A1-GFP distributes to the endoplasmic reticulum, indicating that suberin monomer biosynthesis occurs in this subcellular compartment before intermediates are exported to the apoplast . When designing co-localization experiments, consider that the greatest expression of CYP86A1 occurs in roots, shoots, and leaves, with relatively lower expression in fruits, contrasting with expression patterns of related genes like MYB41, which shows higher expression in fruits .
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How can researchers effectively compare CYP86A1 expression and function across different plant species?
Cross-species studies of CYP86A1 require careful experimental design:
| Aspect | Methodological Approach | Challenges | Solutions |
|---|---|---|---|
| Antibody Cross-Reactivity | Test antibody on multiple species extracts | Epitope conservation varies | Use highly conserved regions as antigens |
| Sequence Homology | Phylogenetic analysis | Identifying true orthologs | Combine sequence similarity with functional testing |
| Functional Conservation | Heterologous expression in model systems | Expression efficiency differences | Use codon-optimized sequences |
| Expression Patterns | Compare tissue-specific expression | Developmental timing differences | Use equivalent developmental stages |
For meaningful cross-species comparisons, researchers should first establish phylogenetic relationships of CYP86A1 homologs. For example, AchnCYP86A1 from kiwifruit (Actinidia chinensis) shows high sequence similarity to Arabidopsis AtCYP86A1 (77%), potato StCYP86A33 (78%), and tobacco NbCYP86A1 (79%) . Functional conservation can be demonstrated through heterologous expression, as shown when AchnCYP86A1 was expressed in N. benthamiana, resulting in increased ω-hydroxyacids with chain lengths C16-C18, similar to the function of AtCYP86A1 .
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What experimental strategies can address functional redundancy when studying CYP86A1?
Addressing functional redundancy in CYP86A1 research requires sophisticated genetic approaches:
Recent studies with MYB transcription factors regulating suberin biosynthesis found that single myb41 mutants showed no visible suberin phenotype despite MYB41's established role in suberization. Further investigation revealed that MYB53, MYB92, and MYB93 were upregulated in the myb41 mutant background, suggesting functional redundancy . Similar redundancy may exist among cytochrome P450 family members affecting CYP86A1 function.
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What are best practices for designing experiments to detect post-translational modifications of CYP86A1?
Studying post-translational modifications of CYP86A1 requires specialized approaches:
| Modification Type | Detection Method | Technical Considerations | Controls |
|---|---|---|---|
| Phosphorylation | Phospho-specific antibodies; Mass spectrometry | Enrichment may be necessary | λ-phosphatase treatment as negative control |
| Glycosylation | Glycosylation-specific stains; Lectin blotting | Deglycosylation enzymes can confirm | PNGase F treatment |
| Ubiquitination | Co-IP with ubiquitin antibodies | Proteasome inhibitors improve detection | K48R mutants as negative controls |
| Membrane Association | Membrane fractionation | Careful preparation of microsomal fractions | Known ER membrane proteins as positive controls |
For CYP86A1, its localization to the endoplasmic reticulum suggests it undergoes typical processing for membrane-bound proteins. As a cytochrome P450, CYP86A1 likely requires electron transfer partners for activity, making protein-protein interaction studies valuable. Experiments designed to detect post-translational modifications should include appropriate controls and consider the ER-resident nature of the protein when designing extraction and enrichment protocols.
