CYP71A15 Antibody

What is CYP71A15 and why is it important in research?

CYP71A15 is a member of the cytochrome P450 family of enzymes that plays crucial roles in plant secondary metabolism. Like other CYP enzymes, it catalyzes substrate oxidation and is involved in metabolizing diverse endogenous compounds and xenobiotics. Research on CYP71A15 is important for understanding plant defense mechanisms, metabolic pathways, and potential biotechnological applications. Antibodies against CYP71A15 are essential tools for detecting and studying this enzyme in various experimental contexts, similar to how CYP1A1 antibodies have been instrumental in studying that particular enzyme's role in detoxifying harmful substances and maintaining metabolic homeostasis .

How specific are commercially available CYP71A15 antibodies?

Specificity is a critical consideration for CYP antibodies. High-quality CYP71A15 antibodies should demonstrate minimal cross-reactivity with other CYP family members. When evaluating antibody specificity, researchers should look for validation data similar to what has been established for other CYP antibodies, such as the CYP1B1 antibody which demonstrated no significant cross-reactivity to either human CYP1A1 or human CYP1A2 protein in Western blot analysis . Validated CYP71A15 antibodies should recognize a single protein band of the expected molecular weight (typically in the 50-60 kDa range) in appropriate samples.

What experimental applications are suitable for CYP71A15 antibodies?

CYP71A15 antibodies can be utilized across multiple experimental applications, similar to other CYP antibodies. The table below outlines common applications and their requirements:

Similar to CYP1A1 antibodies, CYP71A15 antibodies can be expected to function in these applications with proper optimization .

How can I validate the specificity of my CYP71A15 antibody for my specific plant species?

Validating antibody specificity for CYP71A15 in your specific plant species is crucial due to potential sequence variations across species. A comprehensive validation approach should include:

• Positive and negative controls: Include samples with known CYP71A15 expression (positive control) and samples where CYP71A15 is absent or knocked down (negative control).

• Recombinant protein testing: Test the antibody against purified recombinant CYP71A15 from your species of interest.

• Western blot analysis: Verify that the antibody detects a single band of the expected molecular weight, similar to the validation approach used for CYP1B1 antibody which recognized a single protein band of approximately 56 kDa .

• Immunoprecipitation followed by mass spectrometry: This can confirm the identity of the immunoprecipitated protein.

• Pre-absorption test: Pre-incubate the antibody with the immunizing peptide or recombinant protein before staining to confirm that the signal is abolished.

• Cross-species reactivity assessment: If working with multiple plant species, validate the antibody's performance across all relevant species.

The validation strategy should be comprehensive and systematically documented to ensure reliable experimental results.

What are the optimal conditions for using CYP71A15 antibodies in immunohistochemistry of plant tissues?

Optimizing immunohistochemistry (IHC) protocols for plant tissues when using CYP71A15 antibodies requires careful consideration of several factors:

• Fixation method: Plant tissues typically require specialized fixation. A comparison of common fixatives:

  • 4% paraformaldehyde: Preserves protein antigenicity but may require extended fixation times for plant tissues

  • FAA (formaldehyde-acetic acid-alcohol): Better penetration of plant tissues with thick cell walls

  • Methacarn (methanol-chloroform-acetic acid): Alternative for preserving both morphology and antigenicity

• Antigen retrieval: Plant cell walls often necessitate enhanced antigen retrieval methods:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0)

  • Enzymatic treatment with cellulase, pectinase, or driselase to break down cell wall components

• Blocking and antibody incubation:

  • Extended blocking (2-3 hours) with 5% normal serum from the secondary antibody host species

  • Addition of 0.1-0.3% Triton X-100 to improve antibody penetration

  • Longer primary antibody incubation (overnight at 4°C to 48 hours) at dilutions starting at 1:100

  • Multiple washing steps with PBS containing 0.1% Tween-20

• Detection system optimization:

  • Brightfield: HRP-conjugated secondary antibodies with DAB substrate

  • Fluorescence: Consider using Alexa Fluor conjugates for reduced autofluorescence interference from plant compounds

• Counterstaining considerations:

  • Toluidine blue for brightfield applications

  • DAPI for nuclear counterstaining in fluorescence applications

  • Careful selection to avoid interfering with natural plant pigments

The optimal protocol will likely require empirical determination for specific plant tissues and should be validated with appropriate controls.

How can I resolve discrepancies between CYP71A15 protein levels detected by Western blot versus enzyme activity assays?

Discrepancies between protein detection and enzymatic activity are common challenges in CYP research and can arise from several factors:

• Post-translational modifications: CYP enzymes undergo various modifications that may affect activity but not antibody recognition.

• Protein conformation: The antibody may recognize epitopes that are differentially exposed in active versus inactive conformations of the enzyme.

• Cofactor availability: CYP enzymes require electron donors and cofactors for activity; their absence in assays would reduce activity without affecting protein detection.

• Substrate specificity: If the activity assay uses suboptimal substrates, activity might be underestimated despite abundant protein.

• Inhibitors present in samples: Endogenous compounds may inhibit enzyme activity without affecting antibody binding.

Resolution strategies include:

• Multiple antibody approach: Use antibodies targeting different epitopes to confirm protein detection.

• Recombinant enzyme calibration: Generate a standard curve relating protein levels to activity using purified recombinant enzyme.

• Native versus denaturing conditions: Compare results from native PAGE with SDS-PAGE to assess protein functionality.

• Correlation analysis: Perform statistical analysis across multiple samples to determine if there's a consistent relationship between detection methods.

• Mass spectrometry: Use targeted proteomics to quantify the enzyme and identify potential modifications affecting activity.

This approach to resolving discrepancies parallels strategies used with other CYP enzymes, including CYP1B1, where immunoprecipitation and immunoinhibition experiments helped establish that the antibody recognizes the nondenatured protein but does not inhibit enzyme activity .

What are the best strategies for optimizing Western blot protocols specifically for CYP71A15 detection?

Optimizing Western blot protocols for CYP71A15 detection requires systematic adjustment of several parameters:

• Sample preparation:

  • Include protease inhibitors to prevent degradation

  • Add reducing agents (e.g., DTT or β-mercaptoethanol) to ensure proper denaturation

  • Heat samples at 95°C for 5 minutes (standard) or try lower temperatures (70-80°C) if aggregation occurs

• Gel percentage and running conditions:

  • Use 10% polyacrylamide gels for optimal resolution of ~50-60 kDa proteins

  • Consider gradient gels (4-15%) for simultaneous detection of multiple proteins

  • Maintain constant voltage (100-120V) to prevent overheating

• Transfer optimization:

  • Semi-dry transfer: 15-25V for 30-45 minutes

  • Wet transfer: 100V for 1 hour or 30V overnight at 4°C

  • Use methanol-free transfer buffer if protein extraction is difficult

• Blocking and antibody incubation:

  • Test both BSA and non-fat dry milk as blocking agents (3-5%)

  • Primary antibody dilutions ranging from 1:500 to 1:5000

  • Incubation at 4°C overnight often yields cleaner results than room temperature

  • Secondary antibody dilutions typically 1:5000 to 1:20000

• Detection system selection:

  • Enhanced chemiluminescence (ECL) for standard detection

  • Fluorescent secondary antibodies for multiplexing and quantification

  • Consider HRP-conjugated primary antibodies to eliminate secondary antibody cross-reactivity

• Troubleshooting common issues:

Following these optimization steps should yield reliable and reproducible Western blot results for CYP71A15 detection.

How can I develop a reliable quantitative ELISA assay for CYP71A15 in plant extracts?

Developing a quantitative ELISA for CYP71A15 in plant extracts requires careful consideration of several factors:

• ELISA format selection:

  • Sandwich ELISA: Requires two antibodies recognizing different epitopes

  • Competitive ELISA: Useful when only one antibody is available

  • Direct ELISA: Simplest but may have higher background in complex samples

• Sample preparation optimization:

  • Extraction buffer composition: Test phosphate, Tris, and HEPES buffers with different pH values (7.0-8.0)

  • Detergent selection: Non-ionic detergents (0.1% Triton X-100 or NP-40) to solubilize membrane-associated CYP71A15

  • Clarification method: Centrifugation (14,000 × g, 15 min) followed by filtration if necessary

  • Plant-specific interfering compounds: Include polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone (PVPP) to remove phenolic compounds

• Antibody pair optimization (for sandwich ELISA):

  • Capture antibody: 1-10 μg/mL in carbonate-bicarbonate buffer (pH 9.6)

  • Detection antibody: Test conjugation with biotin, HRP, or direct enzyme labeling

  • Orientation testing: Try reversing capture and detection antibodies to determine optimal arrangement

• Standard curve preparation:

  • Recombinant CYP71A15 expression and purification

  • Serial dilutions ranging from 0.1-1000 ng/mL

  • Matrix matching: Prepare standards in extract from CYP71A15-deficient tissue

• Protocol optimization:

  • Coating conditions: 4°C overnight versus 37°C for 2 hours

  • Blocking: BSA versus casein-based blockers (2-5%)

  • Sample incubation: 1-4 hours at room temperature or overnight at 4°C

  • Detection system: Substrate selection based on sensitivity requirements (TMB, ABTS, etc.)

• Validation parameters:

  • Limit of detection: Determined as signal of blank + 3× standard deviation

  • Limit of quantification: Signal of blank + 10× standard deviation

  • Linearity: R² > 0.98 across the working range

  • Recovery: Spike known amounts of recombinant protein into samples (acceptable range: 80-120%)

  • Precision: Intra-assay CV < 10%, inter-assay CV < 15%

  • Specificity: Cross-reactivity testing with related CYP proteins

This methodological approach is similar to established protocols for other CYP antibodies, such as those used for CYP7A1 antibody applications in ELISA .

What techniques can I use to co-localize CYP71A15 with other proteins in plant cells?

Co-localization studies of CYP71A15 with other proteins can provide valuable insights into protein-protein interactions and functional relationships. Several techniques are appropriate:

• Dual immunofluorescence microscopy:

  • Requirements: Antibodies from different host species or directly conjugated to different fluorophores

  • Protocol optimization:

    • Sequential versus simultaneous antibody incubation

    • Cross-adsorption of secondary antibodies

    • Careful control of autofluorescence from plant tissues

  • Analysis: Calculate Pearson's or Manders' colocalization coefficients

• Proximity ligation assay (PLA):

  • Advantage: Detects proteins in close proximity (<40 nm) with high sensitivity

  • Protocol:

    • Primary antibodies from different species

    • Secondary antibodies conjugated to oligonucleotides

    • Signal amplification through rolling circle amplification

  • Output: Distinct fluorescent spots indicate protein proximity

• Fluorescence resonance energy transfer (FRET):

  • Requirements: Antibodies conjugated to appropriate donor/acceptor fluorophore pairs

  • Analysis: Measure donor fluorescence lifetime or sensitized emission

  • Advantage: Can provide quantitative data on protein proximity (1-10 nm)

• Bimolecular fluorescence complementation (BiFC):

  • Approach: Express CYP71A15 and partner protein fused to complementary fragments of fluorescent protein

  • Advantage: Direct visualization of interactions in living cells

  • Limitation: Requires genetic manipulation of plant material

• Co-immunoprecipitation followed by microscopy:

  • Strategy: Perform co-IP to verify interaction, then visualize with microscopy

  • Benefit: Combines biochemical validation with spatial information

Similar to approaches used with CYP1A1 antibodies, these techniques require careful optimization of antibody concentrations and appropriate controls to ensure specificity and minimize background .

How can I minimize non-specific binding when using CYP71A15 antibodies in plant tissues?

Non-specific binding is a common challenge when working with plant tissues due to their complex composition and natural compounds that may interfere with antibody specificity. Several strategies can minimize this issue:

• Optimized blocking protocols:

  • Extended blocking times (2-3 hours minimum)

  • Test different blocking agents:

    • 5% normal serum from secondary antibody host

    • 3-5% BSA

    • Commercial plant-specific blockers

    • Combination of milk proteins and BSA (3% each)

  • Include 0.1-0.3% Triton X-100 in blocking solutions

• Antibody optimization:

  • Titrate antibody concentration systematically

  • Pre-adsorb antibody with plant extract from species lacking CYP71A15

  • Consider using purified IgG fraction rather than whole serum

  • Add 0.05-0.1% Tween-20 to antibody dilution buffer

• Sample preparation refinements:

  • More extensive washing between steps (5-6 washes, 10 minutes each)

  • Include 0.05-0.2M NaCl in wash buffers to reduce ionic interactions

  • Apply antigen retrieval methods appropriate for plant tissues

  • Block endogenous peroxidase activity (for HRP-based detection)

  • Treat with avidin/biotin blocking kit if using biotinylated reagents

• Controls for identifying sources of non-specific binding:

  • Secondary antibody only control

  • Isotype control (non-specific IgG from same host species)

  • Pre-immune serum control

  • Peptide competition assay

These approaches parallel strategies that have proven effective with other CYP antibodies, such as the CYP1B1 antibody, which demonstrated high specificity in Western blot analysis when properly optimized .

What are the most common causes of false positive and false negative results with CYP71A15 antibodies?

Understanding potential sources of false results is crucial for accurate interpretation of data. Common causes include:

False Positive Results:

• Cross-reactivity with related proteins:

  • CYP71A15 belongs to a large family with conserved domains

  • Solution: Validate antibody specificity against recombinant proteins or through knockout controls

• Endogenous enzyme activities:

  • Peroxidase activity in plant tissues can convert chromogenic substrates

  • Solution: Include hydrogen peroxide blocking step (3% H₂O₂, 10 minutes)

• Non-specific binding to plant compounds:

  • Phenolics, alkaloids, and other secondary metabolites

  • Solution: Include additives like PVP or PVPP in extraction and washing buffers

• Fc receptor binding:

  • Some plant proteins may bind antibody Fc regions

  • Solution: Use F(ab')₂ fragments or add normal serum from antibody host species

False Negative Results:

• Epitope masking or modification:

  • Post-translational modifications may block antibody binding

  • Solution: Try multiple antibodies targeting different epitopes

• Insufficient antigen retrieval:

  • Plant cell walls and fixation can mask epitopes

  • Solution: Optimize antigen retrieval methods (heat, pH, enzymatic)

• Protein degradation during sample preparation:

  • Plant proteases may degrade target proteins

  • Solution: Use protease inhibitor cocktails optimized for plants

• Low expression levels:

  • CYP71A15 may be expressed at levels below detection threshold

  • Solution: Use more sensitive detection methods or concentrate samples

• Antibody denaturation:

  • Improper storage or handling

  • Solution: Aliquot antibodies, avoid freeze-thaw cycles, verify activity periodically

Similar issues have been documented with other CYP antibodies, emphasizing the importance of thorough validation and proper controls .

How can I adapt immunoprecipitation protocols for CYP71A15 in plant membrane fractions?

Immunoprecipitation (IP) of CYP71A15 from plant membrane fractions presents unique challenges due to the membrane-associated nature of CYP enzymes. A specialized protocol should include:

• Membrane fraction preparation:

  • Homogenize plant tissue in buffer containing 250mM sucrose, 50mM HEPES-KOH (pH 7.5), 5mM EDTA

  • Include plant protease inhibitor cocktail

  • Differential centrifugation: 10,000 × g (15 min) to remove debris, then 100,000 × g (1 hour) to collect microsomes

  • Resuspend microsomes in solubilization buffer

• Membrane protein solubilization:

  • Test detergent panel:

    • Digitonin (0.5-1%): Milder solubilization preserving protein-protein interactions

    • CHAPS (0.5-1%): Good for maintaining native protein conformation

    • n-Dodecyl β-D-maltoside (0.5-1%): Effective for membrane proteins

    • NP-40 or Triton X-100 (0.5-1%): More stringent conditions

  • Include 150-300mM NaCl to reduce non-specific interactions

  • Solubilize for 1-2 hours at 4°C with gentle rotation

• Pre-clearing optimization:

  • Incubate lysate with protein A/G beads for 1 hour

  • Add 1-5% BSA or 5-10% normal serum from antibody host species

  • Consider using plant-specific pre-clearing agents

• Antibody binding conditions:

  • Use 2-5 μg antibody per 500 μg total protein

  • Extended incubation (overnight at 4°C)

  • Direct antibody conjugation to beads may improve results

• Washing stringency balance:

  • Initial washes: Milder conditions to preserve interactions

  • Final washes: More stringent to reduce background

  • Typical progression: IP buffer → IP buffer + 150mM NaCl → IP buffer + 300mM NaCl

• Elution methods:

  • Denaturing: SDS sample buffer at 70°C (avoid higher temperatures)

  • Native: Antibody-competing peptide elution

  • Acid elution: Glycine buffer (pH 2.5) followed by immediate neutralization

This specialized approach builds upon techniques validated for other CYP family members, such as CYP1B1, where immunoprecipitation was successfully used to study the native protein .

How can I design a multiplex assay to simultaneously detect CYP71A15 and related enzymes in a metabolic pathway?

Designing a multiplex assay for CYP71A15 and related enzymes requires careful selection of detection methods and antibodies:

• Multiplex Western blotting strategies:

  • Size-based separation: Select target proteins with sufficient molecular weight differences

  • Fluorescent detection: Use secondary antibodies conjugated to different fluorophores (e.g., Alexa Fluor 488, 546, 647)

  • Host species variation: Select primary antibodies from different host species

  • Stripping and reprobing: Sequential detection if antibodies cannot be multiplexed

• Multiplex immunofluorescence approaches:

  • Traditional: Limited to 4-5 targets using standard fluorophores

  • Advanced methods:

    • Tyramide signal amplification: Sequential detection of up to 10 targets

    • Spectral unmixing: Distinguishes overlapping fluorophore spectra

    • Iterative bleaching and restaining

  • Controls: Single-stain controls to establish spectral profiles

• Flow cytometry adaptation for plant protoplasts:

  • Protoplast preparation optimization

  • Fixation and permeabilization conditions

  • Compensation matrix development

  • Gating strategy based on cell size and viability

• Mass cytometry (CyTOF) for highly multiplexed detection:

  • Metal-conjugated antibodies eliminate spectral overlap issues

  • Allows simultaneous detection of 40+ targets

  • Requires specialized equipment and antibody conjugation

• Multiplex ELISA formats:

  • Spatially separated: Multi-spot plates

  • Bead-based: Different bead populations coupled to capture antibodies

  • Detection: Differentially labeled detection antibodies or reporter systems

This multiplex approach requires thorough validation to ensure that each target is specifically detected without cross-reactivity, similar to the validation performed for the CYP1B1 antibody which demonstrated no significant cross-reactivity to related CYP proteins .

What considerations are important when designing experiments to study post-translational modifications of CYP71A15?

Studying post-translational modifications (PTMs) of CYP71A15 requires specialized experimental approaches:

• Common PTMs affecting CYP enzymes:

  • Phosphorylation: Affects enzyme activity and protein-protein interactions

  • Glycosylation: May influence protein stability and localization

  • Ubiquitination: Regulates protein degradation

  • Acetylation: Can modulate enzyme function

  • Proteolytic processing: Activation or inactivation

• Sample preparation considerations:

  • Phosphatase inhibitors: Include sodium fluoride (10mM), sodium orthovanadate (1mM), and β-glycerophosphate (10mM)

  • Deubiquitinase inhibitors: N-ethylmaleimide (5mM)

  • Deacetylase inhibitors: Nicotinamide (5mM), trichostatin A (1μM)

  • Lysis conditions: Optimize to preserve modifications of interest

• Detection strategies:

  • PTM-specific antibodies: Anti-phospho, anti-ubiquitin, anti-acetyl lysine

  • Enrichment methods:

    • Phosphopeptides: IMAC, titanium dioxide, phospho-antibody

    • Glycopeptides: Lectin affinity, hydrazide chemistry

    • Ubiquitinated proteins: Ubiquitin-binding domains, anti-ubiquitin antibodies

  • Mass spectrometry approaches:

    • Shotgun proteomics with PTM-specific enrichment

    • Targeted approaches (PRM/MRM) for specific sites

    • Top-down proteomics for intact protein analysis

• Functional validation experiments:

  • Site-directed mutagenesis of modified residues

  • In vitro enzymatic assays comparing modified and unmodified forms

  • Subcellular localization studies before and after stimuli that induce modifications

  • Protein-protein interaction studies comparing modified and unmodified forms

• Considerations specific to plant systems:

  • Plant-specific PTM patterns may differ from animal systems

  • Cell wall interference with extraction efficiency

  • Plant-specific enrichment protocols may be required

This systematic approach to studying PTMs is essential for understanding the regulatory mechanisms affecting CYP71A15 function in plant metabolism.

How can I design experiments to investigate the interaction of CYP71A15 with other proteins in metabolic complexes?

Investigating protein-protein interactions involving CYP71A15 requires a multi-technique approach:

• Co-immunoprecipitation (Co-IP) strategies:

  • Forward and reverse Co-IP to confirm interactions

  • Crosslinking before lysis to capture transient interactions

  • Native versus denaturing conditions

  • Detergent selection critical for membrane-associated complexes

  • Controls: Non-specific IgG, extract from tissues lacking one partner

• Proximity-based labeling approaches:

  • BioID: Fusion of biotin ligase to CYP71A15

  • APEX2: Peroxidase-based proximity labeling

  • Advantages: Captures weak or transient interactions

  • Implementation in plant systems requires optimization of expression systems

• Fluorescence-based interaction studies:

  • FRET: Requires fluorescent protein fusions to potential partners

  • BiFC: Visualizes interactions through complementation of split fluorescent protein

  • FLIM-FRET: Measures fluorescence lifetime changes upon interaction

  • Controls: Non-interacting protein pairs, competition with unlabeled proteins

• Yeast two-hybrid adaptations:

  • Split-ubiquitin system for membrane proteins

  • Systematic screening against cDNA libraries

  • Verification of positive hits with alternative methods

  • Limitations with transmembrane proteins require specialized approaches

• Mass spectrometry-based interactomics:

  • Label-free quantification comparing specific versus control IPs

  • SILAC or TMT labeling for quantitative comparison

  • Crosslinking mass spectrometry (XL-MS) to map interaction interfaces

  • Data analysis: Significance analysis comparing experimental and control samples

• Functional validation of interactions:

  • Mutational analysis of interaction interfaces

  • Competition assays with peptides or small molecules

  • Enzymatic assays in presence/absence of interaction partners

  • In vivo studies using mutants lacking potential interaction partners

This comprehensive approach builds upon established methods for studying protein interactions in other CYP enzymes, such as those used to investigate CYP1B1 protein-protein interactions .