CYP86B1 Antibody

What is CYP86B1 and what role does it play in plant biology?

CYP86B1 is a cytochrome P450 enzyme that functions as a key regulator in suberin biosynthesis in plants. It specifically catalyzes the ω-hydroxylation of very long chain fatty acids (VLCFAs), particularly C22 and C24 fatty acids, which are essential precursors for suberin polyester formation. Suberin is a complex biopolymer that forms protective barriers in plant tissues, particularly in the endodermis of roots and the seed coat .

Functional studies using cyp86B1 mutants (also termed "root aliphatic plant hydroxylase" lines) have demonstrated that disruption of this gene results in altered composition of C22- and C24-hydroxyacids and α,ω-dicarboxylic acids in both root and seed coat aliphatic polyesters . This indicates CYP86B1's essential role in plant barrier formation and protection against environmental stresses.

Where is CYP86B1 expressed in plants and how is it regulated?

CYP86B1 shows high expression in roots and elevated expression in developing seeds. Gene expression profiling indicates that CYP86B1 is co-regulated with CYP86A1, suggesting involvement in the same metabolic pathway . β-glucuronidase (GUS) reporter assays have demonstrated strong CYP86B1 promoter activity at known sites of suberin production, particularly in the endodermis .

Promoter analysis of CYP86B1 has revealed putative cis-acting regulatory elements that may influence its expression patterns. While specific data for CYP86B1 stress responses isn't detailed in the search results, related cytochrome P450s like CYP94B1 show increased expression under salt stress, with expression peaking approximately 4-fold after 6 hours of salt treatment in leaves .

What is the subcellular localization of CYP86B1?

The subcellular localization of CYP86B1 appears to be complex and potentially dependent on experimental approaches. Some research suggests that CYP86B1 contains a putative plastid-targeting N-terminal peptide sequence, with 18% Ser or Thr residues in its N-terminal region, suggesting possible chloroplast localization .

One study using 35S-labeled proteins and antibodies has reported that CYP86B1 is localized in the outer envelope membrane of the chloroplast in pea seedlings . To investigate this further, researchers have constructed fusion proteins with CYP86B1 comprising a C-terminal yellow fluorescent protein (YFP) marker for visualization in transient expression assays .

What approaches should I use to validate a new CYP86B1 antibody?

Proper validation of CYP86B1 antibodies requires a systematic approach:

• Western blot verification:

  • Test against wild-type plant extracts vs. cyp86B1 knockout/knockdown mutants

  • Include recombinant CYP86B1 protein as positive control

  • Verify antibody specificity through peptide competition assays

• Cross-reactivity assessment:

  • Test against related cytochrome P450 family members (especially CYP86A1, which shares ~45% identity)

  • Evaluate specificity across different plant species if working with non-model organisms

• Application-specific validation:

  • For immunolocalization: Compare patterns with previously reported GFP/YFP fusion localization studies

  • For Co-IP studies: Confirm ability to immunoprecipitate native CYP86B1 from plant extracts

The protocol used by researchers in monoclonal antibody development against other proteins, as described in the search results , provides a useful template for validation approaches.

How can I differentiate between CYP86B1 and other closely related cytochrome P450 enzymes?

Distinguishing CYP86B1 from related cytochrome P450 enzymes requires:

• Epitope selection:

  • Generate antibodies against unique regions of CYP86B1 that differ from related P450s

  • Target variable regions rather than conserved catalytic domains

• Genetic verification:

  • Use cyp86B1 knockout/knockdown lines as negative controls

  • Test antibody reactivity against extracts from plants overexpressing specific P450s

• Sequential immunodepletion:

  • Perform immunoprecipitation with antibodies against related P450s before testing for CYP86B1

  • This approach can help confirm specificity in complex samples

• Bioinformatic analysis:

  • Conduct in silico epitope mapping to identify unique regions

  • Use tools like PCPCM (plant cytochrome P450 comparative modeling) for structure prediction

What are the optimal conditions for detecting CYP86B1 via western blotting?

Based on protocols used for similar cytochrome P450 enzymes:

Sample preparation:

• Extract proteins using buffers containing 1-2% non-ionic detergents (e.g., Triton X-100) to solubilize membrane-associated proteins

• Include protease inhibitors to prevent degradation

• Maintain samples at 4°C during extraction

SDS-PAGE conditions:

• Use 10-12% acrylamide gels for optimal resolution of the ~55-60 kDa CYP86B1 protein

• Load 20-30 μg of total protein per lane for standard detection

• Include molecular weight markers (BioRad Precision Plus Protein All Blue Standards or similar)

Transfer and detection:

• Transfer to PVDF membranes (may work better than nitrocellulose for hydrophobic proteins)

• Block with 5% BSA rather than milk (often better for membrane proteins)

• For enhanced sensitivity, consider chemiluminescent detection methods similar to those used for other plant proteins

Controls:

• Include wild-type and cyp86B1 mutant samples

• Consider using recombinant protein with epitope tags as additional controls

How can I optimize immunolocalization of CYP86B1 in plant tissues?

For successful immunolocalization of CYP86B1:

Fixation and embedding:

• For light microscopy: Use 4% paraformaldehyde fixation

• For electron microscopy: Consider 0.5-2% glutaraldehyde

• Vacuum infiltration improves fixative penetration in plant tissues

Antigen retrieval:

• Test heat-induced epitope retrieval (citrate buffer, pH 6.0)

• Enzymatic retrieval with proteinase K may improve accessibility to membrane proteins

Immunostaining protocol:

• Block with 3% BSA, 0.1% Triton X-100 in PBS for 1 hour

• Incubate with primary antibody overnight at 4°C

• Wash 3×15 minutes with PBS + 0.1% Tween-20

• Incubate with fluorescently-labeled secondary antibody for 2 hours

• Wash 3×15 minutes with PBS + 0.1% Tween-20

• Counterstain nuclei with DAPI if needed

• Mount and image

Controls and visualization:

• Include sections from cyp86B1 mutants as negative controls

• Co-stain with established markers for subcellular compartments

• Image using confocal microscopy for optimal resolution

Based on the protocols used for other plant proteins, various monoclonal antibodies show different efficacies in immunostaining, so optimization may be required .

What approaches can I use to study CYP86B1 interactions with other proteins?

To investigate CYP86B1 protein interactions:

Co-immunoprecipitation (Co-IP):

• Use mild detergents (0.5-1% NP-40 or digitonin) for protein extraction

• Pre-clear lysates with Protein A/G beads to reduce background

• Immobilize CYP86B1 antibody on beads before adding lysate

• Elute bound proteins and analyze by mass spectrometry

Yeast two-hybrid screening:

• Create bait constructs with CYP86B1 (consider removing transmembrane domains)

• Screen against cDNA libraries from tissues where CYP86B1 is expressed

• Verify interactions using alternative methods

Bimolecular Fluorescence Complementation (BiFC):

• Generate fusion constructs of CYP86B1 and candidate interactors with split fluorescent protein fragments

• Express in plant protoplasts or via agro-infiltration

• Monitor for fluorescence reconstitution indicating protein interaction

Proximity-dependent labeling:

• Fuse CYP86B1 to BioID or APEX2

• Express in plants to biotinylate proximal proteins

• Purify biotinylated proteins and identify by mass spectrometry

How can I investigate the functional relationship between CYP86B1 and other suberin biosynthesis enzymes?

Genetic approaches:

• Generate double/triple mutants with genes like CYP86A1, GPAT5, FAR5, and RWP1

• Perform complementation studies by expressing one gene in the mutant background of another

• Create chimeric proteins to identify functional domains

Biochemical analysis:

• Perform in vitro enzyme assays with purified proteins to determine substrate preferences

• Analyze metabolite accumulation in various mutant backgrounds

• Use isotope-labeled precursors to track metabolic flux

Expression correlation analysis:

• Generate co-expression networks including genes like CYP86A1, GPAT5, FAR5, RWP1, MYB93 and GDSL-lipase/esterase

• Identify transcription factors that coordinately regulate these genes

• Use ChIP-seq to map binding sites of candidate transcription factors

Protein-protein interaction studies:

• Investigate direct interactions between suberin biosynthetic enzymes

• Map potential metabolon formation through BiFC or FRET analysis

• Study subcellular co-localization of the enzyme complex

What techniques can I use to study the impact of stress on CYP86B1 expression and function?

Expression analysis under stress conditions:

• Perform qRT-PCR to quantify expression changes under various stresses

• Generate and analyze pCYP86B1::GUS reporter lines under different conditions

• Conduct RNA-seq to identify co-regulated genes under stress

Promoter analysis:

• Perform chromatin immunoprecipitation (ChIP) to identify transcription factors binding to the CYP86B1 promoter

• Use yeast one-hybrid assays to screen for stress-responsive transcription factors

• Create promoter deletion constructs to map stress-responsive elements

Protein modification and activity:

• Analyze post-translational modifications under stress conditions

• Measure enzyme activity in microsomes isolated from stressed plants

• Study protein stability and turnover rates during stress

Phenotypic analysis:

• Compare stress sensitivity of wild-type vs. cyp86B1 mutants

• Analyze barrier properties and suberin composition under stress

• Perform complementation studies with stress-induced variants

How can I study posttranslational modifications of CYP86B1?

Identification of modifications:

• Immunoprecipitate CYP86B1 and analyze by mass spectrometry

• Use phospho-specific antibodies to detect phosphorylation events

• Apply Phos-tag gels to separate phosphorylated from non-phosphorylated forms

Modification site analysis:

• Generate site-directed mutants of predicted modification sites

• Test mutant proteins for altered activity, localization, or stability

• Perform complementation studies with modification-site mutants

Regulatory enzymes:

• Identify kinases/phosphatases acting on CYP86B1 through inhibitor studies

• Perform in vitro modification assays with purified enzymes

• Test for direct interactions between CYP86B1 and modifying enzymes

Functional significance:

• Compare activity of modified vs. unmodified forms

• Analyze how modifications affect protein-protein interactions

• Study temporal dynamics of modifications during development or stress

Why am I detecting multiple bands when using CYP86B1 antibody in western blots?

Multiple bands on western blots could result from:

Post-translational processing:

• N-terminal processing for chloroplast targeting (if CYP86B1 contains a chloroplast targeting peptide)

• Different phosphorylation states affecting migration

Technical factors:

• Incomplete denaturation causing aggregation

• Proteolytic degradation during sample preparation

• Non-specific antibody binding to related P450 enzymes

Solutions:

• Include protease inhibitor cocktail in extraction buffer

• Optimize sample denaturation conditions (temperature, SDS concentration)

• Perform peptide competition assays to confirm specificity

• Compare band patterns between wild-type and cyp86B1 mutant tissues

• Use recombinant CYP86B1 with defined size as positive control

Why do I observe variable immunostaining patterns with CYP86B1 antibody in different tissues?

Variable immunostaining could result from:

Biological factors:

• Tissue-specific expression levels of CYP86B1

• Different subcellular localization in specific cell types

• Developmental regulation of expression

• Epitope masking through protein-protein interactions

Technical factors:

• Differential fixative penetration in various tissues

• Tissue-specific autofluorescence

• Varying accessibility of the epitope

Verification approaches:

• Compare with pCYP86B1::GUS or pCYP86B1::GFP expression patterns

• Perform qRT-PCR to confirm expression in different tissues

• Test alternative fixation and permeabilization methods

• Use tyramide signal amplification for low-abundance detection

How can I improve specificity when detecting CYP86B1 in plant extracts with high background?

To improve specificity and reduce background:

Antibody optimization:

• Titrate antibody concentration to determine optimal working dilution

• Pre-absorb antibody with extracts from cyp86B1 mutant plants

• Purify antibody using antigen-affinity chromatography

Sample preparation:

• Use subcellular fractionation to enrich for membrane proteins

• Apply differential centrifugation to isolate specific organelles

• Include competing proteins (BSA, non-fat milk) in blocking solutions

Detection strategies:

• Use monoclonal rather than polyclonal antibodies for higher specificity

• Apply two-color detection with antibodies against different epitopes

• Consider proximity ligation assay for improved specificity

Control experiments:

• Always include cyp86B1 mutant samples as negative controls

• Compare results with alternative detection methods (e.g., mass spectrometry)

• Verify results using epitope-tagged CYP86B1 expressed in plants

Table 1: Comparison of Methods for CYP86B1 Detection

Table 2: Reported Expression Changes of CYP86B1 and Related Genes