CYP71B10 Antibody

Basic Research Questions

• What is CYP71B10 and what is its functional role in Arabidopsis thaliana?

  CYP71B10 is a member of the cytochrome P450 superfamily found in Arabidopsis thaliana, a widely used model plant for studying genetic, cellular, molecular, and developmental biology of flowering plants . It belongs to the CYP71 clan, which is one of the largest gene families in plants . The CYP71 clan has evolved through intensive gene duplication and diversification, with a major evolutionary burst in Angiosperms .

  Functionally, as a cytochrome P450 monooxygenase, CYP71B10 likely catalyzes oxidative reactions in metabolism. While the specific function of CYP71B10 is not explicitly detailed in current literature, members of the CYP71 clan are generally involved in:

  • Biosynthesis of secondary metabolites

  • Plant hormone synthesis pathways

  • Detoxification mechanisms

  • Biopolymer formation

  Interestingly, research has identified a fusion protein of CYP71B10 with eight pentatricopeptide repeat motifs , suggesting potential regulatory functions or specialized protein-protein interactions unique to this enzyme.

• What are the primary applications of CYP71B10 antibodies in plant research?

  Based on the available research data, CYP71B10 antibodies have several important applications in plant research, with Western blotting being the most commonly reported technique .

  Application	Technical Details	Research Value	Sample Preparation Considerations
  Western Blotting (WB)	Detects specific protein expression, determines molecular weight	Quantifies protein abundance changes across conditions	Requires effective protein extraction protocols for plant tissues
  Immunohistochemistry (IHC)	Visualizes tissue and cellular distribution	Provides spatial context for protein expression	Proper fixation critical for epitope preservation
  Immunofluorescence (IF)	High-resolution subcellular localization	Determines organelle association and co-localization	Requires optimization to reduce plant autofluorescence
  Protein Interaction Studies	Co-immunoprecipitation approaches	Identifies interaction partners	Native conditions must be maintained during extraction
  Expression Pattern Analysis	Developmental and stress-responsive studies	Maps CYP71B10 regulation	Consistent sampling across developmental stages

  These applications enable researchers to investigate CYP71B10's role in plant development, stress responses, and metabolic pathway regulation .

• How can I verify the specificity of a CYP71B10 antibody?

  Verifying antibody specificity is critical for ensuring reliable experimental results. The following methodological approach is recommended based on established protocols for antibody validation:

  a) Immunizing peptide blocking experiment:

  • Determine the optimal antibody concentration for your specific application

  • Prepare two identical samples for testing

  • In the "blocked" sample, incubate the antibody with five times excess immunizing peptide by weight

  • Process both samples according to your experimental protocol

  • Compare results - specific signals will be absent or significantly reduced in the blocked sample

  b) Western blot validation:

  • Run positive controls (tissues with known CYP71B10 expression)

  • Include negative controls (tissues without CYP71B10 expression or knockout plants)

  • Verify a single band at the expected molecular weight (~55-60 kDa for most CYP proteins)

  • Check for absence of non-specific bands

  c) Genetic validation:

  • If available, test antibody reactivity in CYP71B10 knockout or knockdown lines

  • The specific signal should be absent or proportionally reduced in these samples

  d) Cross-reactivity assessment:

  • Test against closely related CYP family members, particularly other CYP71B proteins

  • Perform sequence alignment to identify potential cross-reactive epitopes

  • Consider epitope mapping to confirm specificity regions

  These validation steps are essential for distinguishing between highly related cytochrome P450 family members, which often share significant sequence homology .

• What are the optimal sample preparation methods for Western blotting with CYP71B10 antibodies?

  Effective sample preparation is crucial for obtaining reliable results with CYP71B10 antibodies. The following protocol is optimized for plant tissue preparation:

  a) Tissue collection and storage:

  • Harvest young, actively growing tissue when possible (higher protein expression)

  • Flash-freeze in liquid nitrogen immediately after collection

  • Store at -80°C until processing

  • Avoid repeated freeze-thaw cycles

  b) Protein extraction buffer optimization:

  • Use a buffer containing:

    • 50 mM Tris-HCl, pH 7.5

    • 150 mM NaCl

    • 1% Triton X-100 or NP-40

    • 1 mM EDTA

    • Complete protease inhibitor cocktail

    • 1 mM DTT or 2-mercaptoethanol

    • Consider adding 5% glycerol for protein stability

  c) Membrane protein enrichment:

  • Since CYP proteins are membrane-associated, consider microsomal fraction preparation:

    • Homogenize tissue in buffer without detergent

    • Centrifuge at 10,000 × g for 20 minutes to remove debris

    • Ultracentrifuge supernatant at 100,000 × g for 1 hour

    • Resuspend the microsomal pellet in buffer containing 0.1% detergent

  d) SDS-PAGE considerations:

  • Use 10-12% acrylamide gels for optimal separation

  • Load 20-50 μg of total protein per lane

  • Include molecular weight markers

  • Consider including recombinant CYP protein as positive control

  e) Transfer and detection:

  • Transfer to PVDF membrane (better protein retention than nitrocellulose)

  • Verify transfer with reversible staining (Ponceau S)

  • Block with 5% non-fat dry milk in TBST

  • Typical antibody dilution range: 1:500-1:2000

  • Detect with appropriate secondary antibody and visualization system

  This optimized protocol accounts for the membrane-associated nature of cytochrome P450 proteins and maximizes detection sensitivity.

• How do I troubleshoot non-specific binding when using CYP71B10 antibodies?

  Non-specific binding is a common challenge when working with antibodies against plant proteins. The following troubleshooting strategies are recommended:

  a) Optimize blocking conditions:

  • Test different blocking agents:

    • 5% non-fat dry milk in TBST (standard)

    • 3-5% BSA in TBST (may reduce background for some antibodies)

    • Commercial blocking reagents (optimized formulations)

  • Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Add 0.1-0.3% Tween-20 to reduce hydrophobic interactions

  b) Antibody dilution optimization:

  • Test a range of primary antibody dilutions (start with manufacturer's recommendation)

  • Perform a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

  • Extend washing steps (5 washes × 5 minutes each)

  • Reduce secondary antibody concentration if background remains high

  c) Peptide competition controls:

  • Perform blocking peptide experiments to identify specific bands

  • Pre-adsorb antibody with plant extracts from tissues not expressing the target

  • Use IgG isotype control to identify non-specific binding

  d) Buffer and protocol modifications:

  • Increase salt concentration in wash buffers (150 mM to 500 mM NaCl)

  • Add 0.1% SDS to wash buffer for more stringent conditions

  • Reduce antibody incubation temperature (4°C instead of room temperature)

  • Use fresh reagents and prepare buffers with ultrapure water

  e) Sample preparation improvements:

  • Include additional centrifugation steps to remove aggregates

  • Filter samples before loading if necessary

  • Consider immunoprecipitation to enrich for the target protein

  • Add protein extraction modifiers (e.g., PVP or PVPP) to remove plant phenolics

  Persistent non-specific binding may indicate cross-reactivity with related CYP family members, which is a common challenge with plant cytochrome P450 antibodies due to sequence similarity .

Advanced Research Questions

• How can CYP71B10 antibodies be employed in protein-protein interaction studies in Arabidopsis signaling pathways?

  CYP71B10 antibodies can be valuable tools for investigating protein-protein interactions through multiple complementary approaches:

  a) Co-immunoprecipitation (Co-IP) strategy:

  • Prepare plant tissue extracts under non-denaturing conditions

  • Incubate with CYP71B10 antibody coupled to protein A/G beads

  • After washing, elute bound proteins and analyze by:

    • Mass spectrometry for unbiased interactome mapping

    • Western blotting for targeted verification of suspected interactions

  • Critical controls include:

    • IgG isotype control

    • Input sample (pre-immunoprecipitation)

    • Validation in CYP71B10 knockout/knockdown plants

  b) Proximity ligation assay (PLA) for in situ detection:

  • This technique provides single-molecule resolution of protein interactions

  • Fix and permeabilize plant tissues

  • Incubate with CYP71B10 antibody and antibody against potential interaction partner

  • Add PLA probes with complementary oligonucleotides

  • Amplify signal by rolling circle amplification when proteins are in proximity

  • Detect fluorescent signal by confocal microscopy

  • This approach is particularly valuable for transient or weak interactions

  c) Bimolecular Fluorescence Complementation (BiFC) validation:

  • After identifying potential interactors through Co-IP

  • Generate fusion constructs between CYP71B10 and one half of a fluorescent protein

  • Generate fusion constructs between potential interactor and complementary half

  • Co-express in plant cells and analyze by fluorescence microscopy

  • CYP71B10 antibodies can confirm expression levels of fusion proteins

  d) Sequential ChIP for transcriptional complexes:

  • If CYP71B10 is associated with transcriptional regulation

  • Perform ChIP with CYP71B10 antibody followed by re-ChIP with antibody against suspected complex component

  • Analyze enriched regions by qPCR or sequencing

  Based on research with other plant proteins like ICK/KRP cyclin-dependent kinase inhibitors , these approaches can reveal unknown functions and regulatory mechanisms of CYP71B10 in Arabidopsis.

• What methodological considerations are important when using CYP71B10 antibodies in plant stress response studies?

  Studying CYP71B10's role in plant stress responses requires careful experimental design and methodological considerations:

  a) Experimental design framework:

  • Time-course sampling to capture dynamic responses:

    • Early response (minutes to hours)

    • Intermediate response (hours to days)

    • Late/adaptive response (days to weeks)

  • Multiple stress conditions relevant to CYP function:

    • Abiotic stresses (drought, salt, temperature, UV, heavy metals)

    • Biotic stresses (pathogen infection, herbivory)

    • Chemical stresses (xenobiotics, pesticides)

  • Statistical considerations:

    • Minimum 4-5 biological replicates per condition

    • Appropriate statistical tests (ANOVA with post-hoc tests)

    • Effect size calculations

  b) Technical protocol considerations:

  • Standardized sampling approach:

    • Collect samples at same time of day (control for circadian effects)

    • Use plants at identical developmental stages

    • Define precise stress application parameters

  • Protein extraction modifications:

    • Include phosphatase inhibitors to preserve post-translational modifications

    • Consider native extraction for interaction studies

    • Optimize extraction buffers for stress-treated tissues (may require higher detergent)

  • Western blot analysis:

    • Include multiple loading controls (stress can affect "housekeeping" proteins)

    • Perform replicate blots from independent biological samples

    • Use fluorescent secondary antibodies for more accurate quantification

  c) Integrative analysis framework:

  • Combine protein expression data with:

    • Transcriptional analysis (RT-qPCR or RNA-seq)

    • Enzymatic activity assays

    • Metabolite profiling related to CYP71B10 function

    • Phenotypic assays for stress tolerance

  • Data integration methods:

    • Correlation analysis

    • Principal component analysis

    • Pathway mapping

    • Network analysis

  d) Genetic validation approaches:

  • Test CYP71B10 knockout/knockdown plants for altered stress responses

  • Complementation studies to confirm phenotypes are due to CYP71B10 loss

  • Overexpression studies to determine if enhanced CYP71B10 levels alter stress tolerance

  These methodological considerations ensure rigorous investigation of CYP71B10's role in plant stress responses while minimizing experimental artifacts.

• How can evolutionary conservation of CYP71B10 be investigated across plant species using antibody-based approaches?

  Investigating the evolutionary conservation of CYP71B10 across plant species requires specialized approaches:

  a) Cross-species reactivity assessment:

  • Test the CYP71B10 antibody against protein extracts from:

    • Close relatives within Brassicaceae

    • More distant dicot species

    • Monocot species

    • Lower plants (ferns, mosses) if applicable

  • Document cross-reactivity profiles:

    • Band molecular weight

    • Signal intensity

    • Multiple band patterns (potential paralogs)

  • Correlate findings with sequence conservation analysis

  b) Epitope mapping and conservation analysis:

  • Determine the exact epitope recognized by the antibody using:

    • Peptide arrays

    • Deletion mutants

    • Point mutations in recombinant proteins

  • Analyze epitope conservation across species:

    • Sequence alignment of CYP71B10 orthologs

    • Structural modeling of epitope regions

    • Correlation of epitope conservation with antibody reactivity

  c) Comparative immunolocalization:

  • Perform immunohistochemistry or immunofluorescence across species

  • Compare:

    • Tissue expression patterns

    • Subcellular localization

    • Developmental regulation

    • Stress responsiveness

  • Correlate localization differences with functional divergence

  d) Functional conservation analysis:

  • Immunoprecipitate CYP71B10 orthologs from different species

  • Compare:

    • Interaction partners (by mass spectrometry)

    • Post-translational modifications

    • Enzymatic activities

  • Relate findings to metabolic profiles

  This approach is supported by research on the CYP71 clan evolution, which has undergone significant diversification during plant evolution . The CYP71 clan appears to have originated early in plant evolution, with significant expansion in flowering plants, making it an excellent subject for evolutionary studies.

• What are the key challenges and methodological solutions in generating specific antibodies against CYP71B10 given high sequence similarity within the cytochrome P450 family?

  Generating specific antibodies against CYP71B10 presents several technical challenges due to the high sequence conservation within the cytochrome P450 family:

  Challenge	Technical Solution	Methodological Approach	Validation Strategy
  High sequence similarity with other CYP71B proteins	Target unique epitopes	Focus on C-terminal regions which show higher variability	Test cross-reactivity against recombinant CYP71B family proteins
  Conserved functional domains	Avoid heme-binding regions	Use bioinformatic analysis to identify divergent sequences	Perform immunizing peptide blocking experiments
  Transmembrane regions	Focus on hydrophilic regions	Use algorithms to predict antigenic and accessible regions	Test antibody in multiple applications (WB, IHC, IP)
  Low immunogenicity of unique regions	Carrier protein conjugation	KLH conjugation to enhance immune response	Verify antibody titer against immunizing peptide vs. whole protein
  Cross-reactivity in plant tissues	Affinity purification	Purify against specific immunizing peptide	Test in knockout lines

  Based on research with cytochrome P450 antibodies, targeting the C-terminus has proven particularly successful for generating specific antibodies . When selecting peptide antigens for CYP71B10, researchers should:

  a) Perform sequence alignment of all CYP71B family members in the target species
  b) Identify regions with minimal conservation, particularly in the C-terminus
  c) Analyze these regions for:

  • Surface accessibility

  • Hydrophilicity

  • Antigenic potential (using prediction algorithms)

  • Minimal post-translational modification sites
    d) Generate peptide-specific antibodies
    e) Validate specificity using multiple approaches including:

  • Peptide competition

  • Testing in knockout/knockdown lines

  • Cross-reactivity assessment with related proteins

  This strategic approach has been successfully applied to other cytochrome P450 family members and represents the current best practice for generating specific antibodies against CYP71B10.

• What advanced protocols can be employed for immunoprecipitation with CYP71B10 antibodies to study post-translational modifications and protein complexes?

  Advanced immunoprecipitation protocols with CYP71B10 antibodies can reveal important insights about post-translational modifications (PTMs) and protein complexes:

  a) Native complex immunoprecipitation protocol:

  • Buffer optimization:

    • Use mild detergents (0.5-1% NP-40, 0.5% digitonin)

    • Include stabilizing agents (5-10% glycerol)

    • Add protease and phosphatase inhibitor cocktails

    • Consider crosslinking agents for transient interactions

  • Pre-clearing strategy:

    • Incubate lysates with protein A/G beads alone

    • Remove non-specific binding proteins before adding antibody

    • Filter lysate through 0.45 μm filter to remove aggregates

  • Antibody coupling:

    • Covalently couple antibody to beads to prevent co-elution

    • Use magnetic beads for gentler handling

    • Consider tandem affinity purification for increased specificity

  b) Post-translational modification analysis:

  • Phosphorylation:

    • Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

    • Analyze immunoprecipitated proteins by:

      • Phospho-specific Western blotting

      • Phos-tag SDS-PAGE for mobility shift

      • Mass spectrometry with phosphopeptide enrichment

  • Ubiquitination:

    • Add deubiquitinase inhibitors (N-ethylmaleimide)

    • Perform immunoprecipitation under denaturing conditions

    • Detect with anti-ubiquitin antibodies or mass spectrometry

  c) Sequential immunoprecipitation for complex analysis:

  • First immunoprecipitation with CYP71B10 antibody

  • Gentle elution with competing peptide

  • Second immunoprecipitation with antibody against suspected interaction partner

  • Analysis of doubly-purified complex

  • This approach significantly reduces background

  d) Mass spectrometry sample preparation:

  • On-bead digestion:

    • Wash immunoprecipitated beads thoroughly

    • Perform tryptic digestion directly on beads

    • Collect peptides for analysis

  • Specialized protocols for membrane proteins:

    • Use complementary proteases (trypsin, chymotrypsin)

    • Consider enhanced extraction techniques for hydrophobic peptides

    • Apply targeted mass spectrometry methods

  These advanced protocols build upon the foundational immunoprecipitation techniques but are optimized for studying the specific challenges of membrane-associated proteins like CYP71B10, which may form part of larger metabolic complexes in plant cells.