CYP76C3 Antibody

What is CYP76C3 and what is its biological significance?

CYP76C3 is a member of the cytochrome P450 CYP76C subfamily, which is specific to Brassicaceae species. This protein belongs to a genomic cluster on chromosome 2 of Arabidopsis thaliana that includes CYP76C1, CYP76C2, and CYP76C4 . The CYP76C subfamily has undergone high rates of gene duplication and loss in Brassicaceae, suggesting its association with species-specific adaptive functions . Functionally, CYP76C3 is involved in the metabolism of monoterpenols and has been specifically linked to linalool metabolism . Gene expression analysis shows that CYP76C3 is predominantly expressed in flowers upon anthesis, indicating a potential role in floral development or defense mechanisms .

What are the basic specifications of commercially available CYP76C3 antibodies?

CYP76C3 antibodies are typically polyclonal antibodies raised in rabbits against recombinant Arabidopsis thaliana CYP76C3 protein . They are provided in liquid form, preserved in a buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 . These antibodies are antigen-affinity purified and designed for research applications such as ELISA and Western Blot . They are specifically reactive against Arabidopsis thaliana and are intended for research use only, not for diagnostic or therapeutic procedures . Standard storage recommendations include keeping the antibody at -20°C or -80°C and avoiding repeated freeze-thaw cycles .

What experimental applications is the CYP76C3 antibody suitable for?

The CYP76C3 antibody has been validated for enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) techniques . These methods allow researchers to detect and quantify CYP76C3 protein in plant tissue samples. For Western blots, the antibody can be used to identify the CYP76C3 protein based on molecular weight and to compare expression levels across different tissue samples or under various treatment conditions. For ELISA, the antibody enables quantitative measurement of CYP76C3 concentrations in tissue extracts. Each application requires specific optimization of antibody dilution, incubation conditions, and detection methods to ensure reliable results.

How should CYP76C3 antibody be stored and handled to maintain its activity?

For optimal preservation of antibody activity, CYP76C3 antibody should be stored at -20°C or preferably -80°C immediately upon receipt . The antibody is typically supplied in a storage buffer containing 50% glycerol, which prevents damage during freezing. Repeated freeze-thaw cycles should be avoided as they can progressively degrade antibody quality . When working with the antibody, it should be thawed on ice and kept cold during handling. For longer experiments, consider aliquoting the stock solution into smaller volumes to minimize freeze-thaw cycles. Always use clean, DNase/RNase-free tubes and pipette tips when handling antibodies to prevent contamination.

How can I validate the specificity of CYP76C3 antibody for my research?

Validating antibody specificity is crucial for reliable research results, especially when working with proteins that have closely related homologs like the CYP76C family. A comprehensive validation approach should include:

• Positive and negative controls: Use protein extracts from wild-type Arabidopsis thaliana (positive control) and cyp76c3 knockout mutants (negative control) to confirm specificity.

• Cross-reactivity assessment: Test the antibody against recombinant proteins of other CYP76C family members (CYP76C1, CYP76C2, CYP76C4, CYP76C5, CYP76C6, CYP76C7) to evaluate potential cross-reactivity, given their sequence similarities and genomic clustering .

• Epitope mapping: If possible, identify the specific epitope recognized by the antibody to predict potential cross-reactivity with other proteins.

• Immunoprecipitation followed by mass spectrometry: This can confirm that the antibody is pulling down the correct protein and identify any non-specific interactions.

• Peptide competition assay: Pre-incubating the antibody with the immunizing peptide should block specific binding in subsequent assays.

What are the optimal experimental conditions for using CYP76C3 antibody in different tissue types?

Optimizing experimental conditions for CYP76C3 antibody use requires consideration of tissue-specific factors:

For Floral Tissues (High Expression):
Since CYP76C3 is predominantly expressed in flowers upon anthesis , these tissues may require lower antibody concentrations (1:1000 - 1:2000) for Western blotting to prevent oversaturation of signal. Extraction buffers should include protease inhibitors to prevent degradation of the target protein.

For Leaf and Silique Tissues (Moderate Expression):
These tissues show lower expression levels and may require higher antibody concentrations (1:500 - 1:1000) and longer incubation times. Consider using signal enhancement methods such as chemiluminescent substrates with extended exposure times.

For Root Tissues (Low Expression):
Given the minimal expression of CYP76C3 in roots (unlike CYP76C4 which is root-specific) , detection may require antibody concentration optimization (1:250 - 1:500), longer incubation periods, and more sensitive detection methods.

General Recommendations:

• Blocking buffer: 5% non-fat dry milk or BSA in TBST

• Primary antibody incubation: Overnight at 4°C with gentle agitation

• Secondary antibody: Anti-rabbit IgG-HRP at 1:5000 - 1:10000

• Extraction buffer optimization: Test different detergents (Triton X-100, NP-40) for efficient membrane protein extraction

How can I distinguish between CYP76C3 and other closely related CYP76C family members in my experiments?

Distinguishing between closely related CYP76C family members requires strategic experimental approaches:

• Comparative Western blotting: Run parallel blots with antibodies specific to different CYP76C family members and compare band patterns and molecular weights. CYP76C family proteins have slightly different molecular weights that may be resolved on high-percentage or gradient gels.

• Two-dimensional gel electrophoresis: This can separate proteins based on both molecular weight and isoelectric point, potentially providing better resolution of closely related CYP76C proteins.

• Immunohistochemistry with tissue specificity controls: Leverage the known differential expression patterns - CYP76C3 is mainly expressed in flowers, CYP76C4 in roots, CYP76C5 and CYP76C7 predominantly in siliques .

• RT-qPCR validation: Complement protein detection with gene expression analysis using highly specific primers for each CYP76C family member to correlate protein and mRNA levels.

• Mass spectrometry: For definitive identification, use immunoprecipitation followed by mass spectrometry to identify peptides unique to CYP76C3.

What strategies can be employed to investigate CYP76C3's role in monoterpenol metabolism using the antibody?

Investigating CYP76C3's role in monoterpenol metabolism requires integration of protein detection with functional analysis:

• Subcellular localization studies: Use the CYP76C3 antibody in immunogold electron microscopy or immunofluorescence to determine the precise subcellular localization of the enzyme, providing insights into its functional context.

• Protein-substrate interaction assays: Combine the antibody with pull-down assays to identify potential binding partners or regulatory proteins that may influence CYP76C3 activity in monoterpenol metabolism.

• Activity correlation studies: Use the antibody to quantify CYP76C3 protein levels in tissues with different monoterpenol contents, analyzing the correlation between protein abundance and metabolite profiles.

• In vitro enzyme assays with immunodepleted extracts: Deplete plant extracts of CYP76C3 using the antibody and compare the monoterpenol metabolism capacity before and after depletion.

• Immunoprecipitation followed by activity assays: Use the antibody to purify native CYP76C3 from plant tissues and assess its activity against various monoterpenol substrates, comparing with the known activities from recombinant expression systems.

• Comparative analysis across CYP76C enzymes: Given that CYP76C1, CYP76C2, and CYP76C4 show different substrate preferences and product profiles for monoterpenols , use the antibody to investigate whether these differences correlate with protein structure or expression patterns.

How can I design experiments to study the potential role of CYP76C3 in herbicide metabolism using the antibody?

Given that some CYP76C family members (CYP76C1, CYP76C2, and CYP76C4) metabolize phenylurea herbicides and confer herbicide tolerance , designing experiments to investigate CYP76C3's role in this process requires:

• Herbicide treatment time-course: Expose plants to phenylurea herbicides and use the CYP76C3 antibody to track protein expression changes over time through Western blotting or ELISA.

• Correlation of protein levels with herbicide resistance: Compare CYP76C3 protein abundance across plant lines with different herbicide tolerance levels to establish potential correlations.

• Co-immunoprecipitation studies: Use the antibody to identify proteins that interact with CYP76C3 in the presence versus absence of herbicides, potentially revealing regulatory mechanisms.

• In vitro herbicide metabolism assays: Immunopurify native CYP76C3 and test its ability to metabolize different herbicides, comparing activity with other CYP76C enzymes known to metabolize phenylureas.

• Transgenic studies with quantitative protein analysis: In plants overexpressing CYP76C3, use the antibody to quantify protein levels and correlate with herbicide tolerance phenotypes. This approach has successfully demonstrated that ectopic expression of CYP76C1, CYP76C2, and CYP76C4 in whole plants conferred herbicide tolerance .

• Protein localization during herbicide stress: Use immunohistochemistry to determine if CYP76C3 subcellular localization changes upon herbicide exposure.

What is the recommended protocol for using CYP76C3 antibody in Western blot analysis?

Recommended Western Blot Protocol for CYP76C3 Detection:

• Sample Preparation:

  • Extract total protein from plant tissue using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail.

  • For membrane proteins like cytochrome P450s, include 1-2% digitonin or 0.5% DDM for efficient extraction.

  • Quantify proteins using Bradford or BCA assay.

• Gel Electrophoresis:

  • Load 20-50 μg of total protein per lane.

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

  • Include positive control (wild-type Arabidopsis flower extract) and negative control (cyp76c3 mutant extract if available).

• Transfer:

  • Transfer proteins to PVDF membrane (preferred over nitrocellulose for hydrophobic proteins).

  • Use semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 1 hour.

• Blocking:

  • Block membrane with 5% non-fat dry milk in TBST (TBS + 0.1% Tween-20) for 1 hour at room temperature.

• Primary Antibody Incubation:

  • Dilute CYP76C3 antibody 1:500 - 1:1000 in blocking buffer.

  • Incubate overnight at 4°C with gentle agitation.

• Washing:

  • Wash membrane 3 times with TBST, 5 minutes each.

• Secondary Antibody Incubation:

  • Incubate with HRP-conjugated anti-rabbit IgG (1:5000 in blocking buffer) for 1 hour at room temperature.

• Detection:

  • Wash 3 times with TBST, 5 minutes each.

  • Develop using enhanced chemiluminescence (ECL) substrate.

  • Expected band size: approximately 55-60 kDa for CYP76C3.

• Optimization Tips:

  • For weak signals, try longer primary antibody incubation or higher antibody concentration.

  • For high background, increase washing time or reduce antibody concentration.

  • When working with flower tissues (high expression), shorter exposure times may be needed.

How can I optimize immunoprecipitation protocols for CYP76C3 research?

Optimized Immunoprecipitation Protocol for CYP76C3:

• Sample Preparation:

  • Homogenize 1-2 g of plant tissue in 3-4 ml of IP buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, protease inhibitor cocktail).

  • For membrane-bound CYP76C3, include 1% digitonin or 0.5% DDM in the buffer.

  • Centrifuge at 14,000 × g for 15 minutes at 4°C and collect the supernatant.

  • Pre-clear lysate with 50 μl of Protein A/G beads for 1 hour at 4°C.

• Antibody Binding:

  • Add 2-5 μg of CYP76C3 antibody to 500 μl of pre-cleared lysate.

  • Incubate overnight at 4°C with gentle rotation.

• Immunoprecipitation:

  • Add 50 μl of Protein A/G beads and incubate for 2-3 hours at 4°C.

  • Collect beads by centrifugation at 1,000 × g for 2 minutes.

  • Wash beads 4 times with IP buffer containing decreasing detergent concentrations.

• Elution Options:

  • For protein analysis: Elute proteins by boiling beads in 50 μl of 2× SDS sample buffer.

  • For activity assays: Use gentle elution with 100 mM glycine (pH 3.0) followed by immediate neutralization.

• Critical Optimization Parameters:

  • Detergent type and concentration: Test different detergents (Triton X-100, CHAPS, digitonin) for optimal CYP76C3 solubilization.

  • Salt concentration: Adjust NaCl concentration (150-300 mM) to reduce non-specific binding.

  • Antibody amount: Titrate antibody concentration to find the optimal ratio for efficient immunoprecipitation.

  • Cross-linking option: Consider cross-linking the antibody to beads for cleaner elution without antibody contamination.

• Validation Controls:

  • Negative control: Perform parallel IP with non-specific rabbit IgG.

  • Input control: Save a small aliquot of pre-cleared lysate.

  • Supernatant control: Save post-IP supernatant to assess depletion efficiency.

What approaches can I use to develop a quantitative ELISA for CYP76C3 protein?

Developing a Quantitative ELISA for CYP76C3 Protein:

• Assay Format Selection:

  • Sandwich ELISA: Requires two antibodies recognizing different epitopes of CYP76C3. If only one antibody is available, consider raising a second antibody against a different epitope or using the rational design approach described in search result .

  • Indirect ELISA: Simpler approach when only one antibody is available. Coat plates with plant extract and detect CYP76C3 with the specific antibody.

• Protocol Development:

  • Coating: For indirect ELISA, coat microplate wells with plant extract (10-50 μg/ml) in carbonate buffer (pH 9.6) overnight at 4°C.

  • Blocking: Block with 3% BSA in PBS for 1 hour at room temperature.

  • Primary antibody: Add CYP76C3 antibody (1:500-1:2000) in blocking buffer, incubate for 2 hours at room temperature.

  • Secondary antibody: Add HRP-conjugated anti-rabbit IgG (1:5000) in blocking buffer, incubate for 1 hour.

  • Detection: Add TMB substrate, develop for 15-30 minutes, stop with 2N H₂SO₄, read absorbance at 450 nm.

• Standard Curve Generation:

  • Express and purify recombinant CYP76C3 protein to use as a standard.

  • Prepare a dilution series (0-1000 ng/ml) to create a standard curve.

  • Include standards on each plate to account for plate-to-plate variation.

• Assay Optimization:

  • Antibody concentration: Titrate primary and secondary antibodies to determine optimal concentrations.

  • Incubation times and temperatures: Test different combinations to improve sensitivity and reduce background.

  • Extraction buffer composition: Optimize for efficient solubilization of membrane-bound CYP76C3.

• Validation Parameters:

  • Specificity: Test against extracts from cyp76c3 knockout plants and recombinant CYP76C family proteins.

  • Sensitivity: Determine limit of detection and quantification.

  • Linearity: Ensure signal is linear within the expected concentration range.

  • Precision: Assess intra- and inter-assay variation (%CV should be <15%).

  • Recovery: Spike samples with known amounts of recombinant CYP76C3 and calculate recovery percentage.

How can I use CYP76C3 antibody to investigate protein-protein interactions in monoterpenol metabolism pathways?

Methods for Investigating CYP76C3 Protein-Protein Interactions:

• Co-Immunoprecipitation (Co-IP):

  • Use CYP76C3 antibody to pull down CYP76C3 and its interacting partners from plant extracts.

  • Analyze co-precipitated proteins by mass spectrometry to identify novel interaction partners.

  • Validation can be performed by reciprocal Co-IP with antibodies against identified partners.

  • This approach can reveal interactions with other enzymes in the monoterpenol metabolism pathway or regulatory proteins.

• Proximity-Dependent Biotin Identification (BioID):

  • Create a fusion protein of CYP76C3 with a biotin ligase.

  • Express the fusion in plants and allow biotin labeling of proximal proteins.

  • Use the CYP76C3 antibody to confirm expression and localization of the fusion protein.

  • Purify biotinylated proteins and identify by mass spectrometry.

  • This method can capture both stable and transient interactions within the native cellular environment.

• Bimolecular Fluorescence Complementation (BiFC):

  • Generate constructs of CYP76C3 and potential interacting partners fused to complementary fragments of a fluorescent protein.

  • Co-express in plant cells and visualize interaction-dependent fluorescence.

  • Use the CYP76C3 antibody in parallel experiments to confirm expression levels of the fusion protein.

  • This approach provides spatial information about where in the cell these interactions occur.

• Pull-Down Assays with Multiple CYP76C Family Members:

  • Given the functional overlap among CYP76C enzymes in monoterpenol metabolism , investigate potential hetero-oligomerization.

  • Express tagged versions of different CYP76C family members.

  • Use CYP76C3 antibody to pull down complexes and analyze the presence of other CYP76C proteins.

  • This can reveal functional complexes that may explain the overlapping yet distinct substrate specificities observed in the CYP76C family.

• Crosslinking-Immunoprecipitation:

  • Treat plant tissues with a protein crosslinker to stabilize transient interactions.

  • Immunoprecipitate with CYP76C3 antibody and analyze complexes by SDS-PAGE and mass spectrometry.

  • This can capture weak or transient interactions that might be missed by standard Co-IP.

What are common issues when working with CYP76C3 antibody and how can they be resolved?

Common Issues and Solutions in CYP76C3 Antibody Applications:

• Weak or No Signal in Western Blots:

  • Possible causes: Low protein expression, inefficient protein extraction, antibody degradation, or improper detection conditions.

  • Solutions:

    • Enrich samples from tissues with known high expression (flowers upon anthesis)

    • Optimize extraction buffer with appropriate detergents for membrane proteins

    • Try longer primary antibody incubation (overnight at 4°C)

    • Increase antibody concentration (1:250 - 1:500)

    • Use more sensitive detection reagents (high-sensitivity ECL)

    • Verify antibody activity with a dot blot of recombinant CYP76C3 protein

• High Background in Immunoassays:

  • Possible causes: Non-specific binding, excessive antibody concentration, inadequate blocking, or cross-reactivity.

  • Solutions:

    • Increase blocking agent concentration (5-10% BSA or milk)

    • Add 0.1-0.5% Tween-20 to washing buffer

    • Titrate antibody to lower concentration

    • Pre-adsorb antibody with plant extract from cyp76c3 knockout plants

    • Increase number and duration of wash steps

    • Try different blocking agents (BSA, casein, or commercial blockers)

• Cross-Reactivity with Other CYP76C Family Members:

  • Possible causes: Epitope similarity between closely related CYP76C proteins.

  • Solutions:

    • Use extracts from tissues with differential expression patterns (e.g., roots for CYP76C4 vs. flowers for CYP76C3)

    • Pre-adsorb antibody with recombinant proteins of other CYP76C family members

    • Perform peptide competition assays to confirm specificity

    • Consider developing a monoclonal antibody targeting unique epitopes

• Inconsistent Immunoprecipitation Results:

  • Possible causes: Insufficient protein solubilization, weak antibody binding, or protein degradation.

  • Solutions:

    • Optimize lysis buffer composition for membrane proteins

    • Increase antibody amount or incubation time

    • Add protease inhibitors freshly before extraction

    • Try different bead types (Protein A, Protein G, or Protein A/G)

    • Cross-link antibody to beads to prevent co-elution

    • Perform all steps at 4°C to minimize protein degradation

How can I validate that my CYP76C3 antibody is still functional after storage?

Validating CYP76C3 Antibody Functionality After Storage:

• Dot Blot Assay:

  • Spot dilutions of recombinant CYP76C3 protein (if available) onto a membrane

  • Process as a regular Western blot with stored antibody

  • Compare signal intensity with results from previous tests or fresh antibody aliquots

  • This provides a quick assessment of binding capacity without gel electrophoresis

• Control Western Blot:

  • Run a small Western blot using a standard positive control sample (Arabidopsis flower extract)

  • Compare band intensity and specificity with previous results

  • Look for expected band at approximately 55-60 kDa

  • Assess background levels as an indicator of antibody quality

• ELISA Titration:

  • Perform a simple indirect ELISA with serial dilutions of the antibody

  • Compare the dose-response curve with previously established curves

  • Calculate the EC50 (half maximal effective concentration) value and compare with historical data

  • Significant changes in EC50 indicate altered antibody functionality

• Functional Parameters to Assess:

  • Titer: The highest dilution that still gives a positive signal above background

  • Specificity: Absence of non-specific bands in Western blot or cross-reactivity in ELISA

  • Sensitivity: Minimum amount of antigen detectable

  • Signal-to-noise ratio: Comparison of specific signal to background

• Storage Optimization Based on Results:

  • If activity is reduced, consider adding stabilizers (1% BSA, 5% glycerol)

  • For partially degraded antibody, use at higher concentration

  • If significant degradation is observed, consider purchasing new antibody

  • Implement improved storage practices (smaller aliquots, lower temperature, avoid freeze-thaw cycles)

How do I establish optimal antibody dilutions for different experimental systems?

Establishing Optimal CYP76C3 Antibody Dilutions:

• Systematic Titration Approach:

  • Prepare a dilution series of the antibody (1:100, 1:500, 1:1000, 1:2000, 1:5000)

  • Test each dilution against a standard positive control sample

  • Evaluate based on signal strength, specificity, and background levels

  • The optimal dilution provides strong specific signal with minimal background

• Application-Specific Considerations:

  For Western Blotting:

  • Start with manufacturer's recommended range (typically 1:500 - 1:1000)

  • Adjust based on protein abundance in different tissues:

    • Flowers (high expression): 1:1000 - 1:2000

    • Leaves/siliques (moderate expression): 1:500 - 1:1000

    • Roots (low expression): 1:250 - 1:500

  • Consider detection method sensitivity (chemiluminescence vs. chromogenic)

  For ELISA:

  • Typically requires higher dilution than Western blotting

  • Start with 1:1000 and test 2-fold dilutions in both directions

  • Plot a standard curve for each dilution to identify the one with best linear range

  For Immunohistochemistry/Immunofluorescence:

  • Start with more concentrated antibody (1:100 - 1:500)

  • Include appropriate negative controls to assess background

  • Optimize blocking conditions in parallel with antibody dilution

• Sample-Specific Optimization:

  • Adjust dilutions based on protein expression levels in different sample types

  • For recombinant protein or overexpression systems, use higher dilutions

  • For native expression in plant tissues, use lower dilutions

  • Consider tissue-specific interfering compounds that may affect antibody binding

• Quantitative Assessment Method:

  • Calculate signal-to-noise ratio for each dilution

  • Determine the dilution that gives maximum signal-to-noise ratio

  • Consider the linear range of detection for quantitative applications

  • Document optimal conditions for different applications and sample types

How can I use CYP76C3 antibody to investigate the evolution of monoterpenol metabolism in Brassicaceae?

Investigating Evolutionary Aspects of CYP76C3 Using Antibody-Based Approaches:

• Cross-Species Immunoreactivity Analysis:

  • Test the CYP76C3 antibody against protein extracts from various Brassicaceae species

  • Compare band patterns, intensities, and apparent molecular weights

  • This can reveal conservation or divergence of the CYP76C3 protein across species

  • The high rates of CYP76C gene duplication and loss in Brassicaceae suggest this might yield interesting evolutionary insights

• Correlation with Genomic Analysis:

  • Combine immunoblot data with genomic information about CYP76C gene clusters

  • Create a presence/absence matrix of immunoreactive bands across species

  • Correlate with phylogenetic relationships and gene duplication/loss events

  • This can reveal how protein expression patterns have evolved alongside gene family expansion

• Functional Conservation Assessment:

  • Immunoprecipitate CYP76C3-like proteins from different Brassicaceae species

  • Test their enzymatic activity against monoterpenol substrates

  • Compare substrate specificity and product profiles across species

  • Analyze whether functional diversification correlates with sequence divergence

• Subcellular Localization Comparison:

  • Use immunolocalization to compare CYP76C3 subcellular distribution across species

  • Changes in localization may indicate functional adaptation during evolution

  • Correlate with differences in monoterpenol metabolism or ecological adaptations

• Tissue-Specific Expression Pattern Analysis:

  • Compare CYP76C3 protein expression patterns across tissues in different Brassicaceae

  • Identify conserved vs. species-specific expression patterns

  • This can reveal evolutionary shifts in the role of CYP76C3 in plant metabolism and development

• Interactome Evolution Study:

  • Use immunoprecipitation to identify CYP76C3 interaction partners across species

  • Compare protein-protein interaction networks

  • Identify core conserved interactions vs. species-specific ones

  • This can reveal how the functional context of CYP76C3 has evolved

What approaches can I use to study post-translational modifications of CYP76C3 protein?

Studying Post-Translational Modifications (PTMs) of CYP76C3:

• Immunoprecipitation Coupled with PTM-Specific Detection:

  • Use CYP76C3 antibody to immunoprecipitate the protein from plant tissues

  • Analyze the precipitated protein with:

    • Phospho-specific staining (Pro-Q Diamond)

    • Glycoprotein staining (Pro-Q Emerald)

    • Ubiquitin/SUMO Western blotting

  • This approach provides initial screening for major PTM types

• Mass Spectrometry Analysis of Immunopurified CYP76C3:

  • Immunoprecipitate CYP76C3 from different tissues or treatment conditions

  • Perform tryptic digestion and analyze by LC-MS/MS

  • Search for mass shifts corresponding to common PTMs:

    • Phosphorylation (+80 Da)

    • Acetylation (+42 Da)

    • Glycosylation (variable mass shifts)

    • Ubiquitination (Gly-Gly remnant after trypsin digestion)

  • Compare PTM profiles across different physiological conditions

• PTM-Specific Antibody Approach:

  • After immunoprecipitation with CYP76C3 antibody, probe with PTM-specific antibodies:

    • Anti-phosphoserine/threonine/tyrosine

    • Anti-acetyllysine

    • Anti-ubiquitin/SUMO

  • This can confirm the presence of specific modifications

• Site-Directed Mutagenesis Validation:

  • Identify potential PTM sites from mass spectrometry data

  • Generate site-directed mutants (e.g., S→A for phosphorylation sites)

  • Express mutant proteins and assess:

    • Changes in enzyme activity

    • Protein stability

    • Subcellular localization

    • Interaction with partner proteins

  • Use the CYP76C3 antibody to confirm expression levels of mutant proteins

• Physiological Relevance Studies:

  • Compare PTM profiles across:

    • Different developmental stages

    • Stress conditions (herbicide exposure, pathogen attack)

    • Tissues with varying monoterpenol content

  • This can reveal how PTMs regulate CYP76C3 function in response to environmental cues

How can I develop a high-throughput screening assay for compounds that modulate CYP76C3 activity using the antibody?

Developing High-Throughput Screening Assays for CYP76C3 Modulators:

• ELISA-Based Activity Modulation Assay:

  • Immobilize CYP76C3 antibody on microplate wells

  • Capture CYP76C3 from plant extracts or using recombinant protein

  • Add potential modulatory compounds along with substrate

  • Detect product formation using:

    • Colorimetric/fluorometric detection of oxidation products

    • Coupled enzyme assays that detect monoterpenol conversion

  • This approach enables screening of thousands of compounds in microplate format

• Thermal Shift Assay (Differential Scanning Fluorimetry):

  • Mix purified CYP76C3 (immunopurified or recombinant) with test compounds

  • Add fluorescent dye that binds to hydrophobic regions (SYPRO Orange)

  • Monitor fluorescence during gradual temperature increase

  • Binding compounds will alter protein thermal stability (Tm)

  • Use CYP76C3 antibody to verify protein quality before assay

  • This assay can rapidly identify compounds that interact directly with CYP76C3

• Cellular Reporter System:

  • Develop a cell-based assay where CYP76C3 activity is linked to a reporter output

  • Options include:

    • Yeast system with growth selection based on monoterpenol detoxification

    • Plant cell system with fluorescent reporters linked to CYP76C3 activity

  • Add test compounds and monitor reporter signal changes

  • Use the CYP76C3 antibody to confirm protein expression in the cellular system

  • This approach identifies modulators that work in cellular context

• Competitive Binding Assay:

  • Develop a competition assay where compounds compete with labeled substrate

  • Options include:

    • Fluorescently labeled substrate analogs

    • Substrate-mimicking affinity probes

  • Detect displacement using fluorescence polarization or other methods

  • Validate hits by analyzing their effects on CYP76C3 enzyme kinetics

  • Use the antibody to confirm that observed effects are specific to CYP76C3

• Data Analysis and Hit Validation:

  • Implement statistical methods for hit identification (Z'-factor, signal-to-background ratio)

  • Establish dose-response relationships for primary hits

  • Classify compounds as activators, inhibitors, or substrate competitors

  • Validate with orthogonal assays including direct enzymatic assays with immunopurified CYP76C3

  • Use computational approaches to identify chemical features associated with activity