CYP82G1 Antibody

What is CYP82G1 and what is its biological function?

CYP82G1 (At3g25180) is a cytochrome P450 monooxygenase of the Arabidopsis CYP82 family responsible for the oxidative cleavage of the C20-precursor (E,E)-geranyllinalool to produce the C16-homoterpene (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) . This enzyme also converts the C15-analog (E)-nerolidol to the C11-homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) .

Biologically, CYP82G1 contributes to indirect plant defense mechanisms. The homoterpene volatiles produced by CYP82G1 are released from herbivore-damaged tissue and attract natural enemies of herbivorous pests . These volatile compounds are also commonly emitted from night-scented flowers and can induce defensive responses in plant-plant interactions, functioning as important signaling molecules .

How do I express and purify recombinant CYP82G1 for antibody production?

For successful recombinant CYP82G1 expression and purification, follow this methodological approach:

• Vector Selection: Use Gateway-compatible expression vectors such as pHIS9 with an N-terminal His-tag for efficient purification . Alternative vectors like pCRT7/CT-TOPO TA have also proven effective for CYP82G1 expression .

• Expression System: Transform Escherichia coli with your expression construct. Grow transformed cells in LB medium at optimal conditions (typically 37°C until OD600 reaches 0.6-0.8) .

• Induction Protocol: Induce protein expression with isopropylthio-β-galactoside (IPTG) at appropriate concentrations (0.1-1.0 mM). For membrane-associated proteins like cytochrome P450s, consider reduced temperature (16-25°C) during the induction phase .

• Purification Steps:

  • Harvest cells by centrifugation

  • Lyse cells using appropriate buffer containing protease inhibitors

  • Purify using Ni-NTA affinity chromatography for His-tagged proteins

  • Consider additional purification steps if needed (ion exchange, gel filtration)

• Quality Control: Verify purified protein integrity via SDS-PAGE and Western blotting before antibody production .

What are the recommended strategies for validating CYP82G1 antibody specificity?

Robust validation of CYP82G1 antibodies requires multiple complementary approaches:

• Western Blot Analysis: Test antibody against wild-type plants and cyp82g1 knockout mutants. A specific antibody will detect a band of approximately 55-60 kDa in wild-type samples that should be absent in knockout mutants .

• Immunoprecipitation: Perform immunoprecipitation followed by mass spectrometry to confirm the precipitated protein is indeed CYP82G1 .

• Transgenic Controls: Use plants overexpressing tagged CYP82G1 (e.g., 35S:Flag-CYP82G1) as positive controls, similar to approaches used for other proteins like HDA6 .

• Cross-Reactivity Assessment: Test against recombinant CYP82G1 and closely related CYP82 family members to ensure specificity within this enzyme family.

• Immunolocalization: Perform immunofluorescence or immunogold labeling to verify that the antibody localizes to expected subcellular compartments where CYP82G1 functions.

How can CYP82G1 antibodies be applied in chromatin immunoprecipitation (ChIP) studies?

While CYP82G1 itself is not directly associated with chromatin, researchers interested in studying potential transcriptional regulation mechanisms might employ ChIP methodologies with appropriate modifications:

• Sample Preparation: Harvest plant tissue (preferably at times of high CYP82G1 expression, such as after herbivore damage) and cross-link protein-DNA complexes using formaldehyde (1-1.5% for 10-15 minutes) .

• Chromatin Shearing: Sonicate using a Bioruptor Pico or Covaris E220 system to achieve optimal chromatin fragmentation (200-500 bp fragments) . Verify shearing efficiency via agarose gel electrophoresis.

• Immunoprecipitation Protocol: Incubate sheared chromatin with anti-CYP82G1 antibody bound to protein A/G magnetic beads. Include appropriate controls (IgG, input samples) .

• Analysis Options:

  • ChIP-qPCR for targeted regions of interest

  • ChIP-seq for genome-wide binding profile analysis

• Data Interpretation: Focus on analyzing promoter regions of genes co-expressed with CYP82G1 or involved in terpene biosynthesis pathways to identify potential regulatory elements .

What experimental approaches can detect changes in CYP82G1 expression and activity during plant stress responses?

To comprehensively assess CYP82G1 regulation during stress responses, implement the following multifaceted approach:

• Transcriptional Analysis:

  • RT-qPCR to quantify CYP82G1 mRNA levels in response to herbivory or other stresses

  • RNA-seq for genome-wide expression profiling

  • Analyze expression patterns throughout the day as circadian regulation may influence expression

• Protein Level Assessment:

  • Western blotting with anti-CYP82G1 antibody to quantify protein abundance

  • Study protein stability using cycloheximide chase assays

• Activity Assays:

  • GC-MS analysis to quantify TMTT production as a direct measure of CYP82G1 activity

  • In vitro enzyme assays using purified recombinant CYP82G1 with (E,E)-geranyllinalool substrate

• Localization Studies:

  • Immunolocalization to track potential changes in subcellular distribution during stress

  • Co-localization with stress-responsive organelles or proteins

• Post-translational Modifications:

  • Immunoprecipitate CYP82G1 followed by LC-MS/MS to identify potential modifications like phosphorylation or nitrosylation that may regulate activity

How can I design experiments to elucidate the structure-function relationship of CYP82G1?

A comprehensive structure-function analysis of CYP82G1 requires integration of computational, biochemical, and genetic approaches:

• Homology Modeling and Substrate Docking:

  • Generate a structural model based on crystallized P450 structures

  • Perform substrate docking simulations to predict binding modes of (E,E)-geranyllinalool and (E)-nerolidol

  • The mechanism appears to involve oxidative bond cleavage via syn-elimination of the polar head, together with an allylic C-5 hydrogen atom

• Site-Directed Mutagenesis:

  • Target residues predicted to be important for substrate binding and catalysis

  • Focus on the active site heme-binding region and substrate recognition sites

  • Use PCR-based mutagenesis approaches with specific cycling conditions as described in methodology references :

    Cycle Step	Temperature	Time	Cycles
    Initial denaturation	95°C	2 min	1
    Denaturation	95°C	30 sec	18
    Annealing	55°C	1 min	18
    Extension	72°C	7 min	18
    Final extension	72°C	10 min	1

• Enzymatic Characterization:

  • Express and purify mutant variants

  • Determine kinetic parameters (Km, Vmax, kcat) for each variant

  • Compare catalytic efficiencies (kcat/Km) between wild-type and mutant enzymes

• In Planta Validation:

  • Complement cyp82g1 knockout plants with wild-type or mutant variants

  • Quantify TMTT production to assess functional complementation

  • Perform herbivore response assays to determine biological significance

What protocols are recommended for analyzing CYP82G1-substrate interactions using advanced analytical techniques?

For rigorous characterization of CYP82G1-substrate interactions, implement these analytical approaches:

• Isothermal Titration Calorimetry (ITC):

  • Directly measure binding affinity and thermodynamic parameters

  • Use purified CYP82G1 protein and chemically synthesized substrates

  • Analyze data to determine KD, ΔH, ΔS, and stoichiometry

• Surface Plasmon Resonance (SPR):

  • Immobilize CYP82G1 on a sensor chip

  • Flow substrate solutions at varying concentrations

  • Determine kon and koff rates and calculate binding affinity

• Mass Spectrometry-Based Approaches:

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to map conformational changes upon substrate binding

  • LC-MS/MS analysis to identify reaction intermediates and products

  • Use stable isotope-labeled substrates to track reaction progression

• Spectroscopic Methods:

  • UV-visible spectroscopy to monitor characteristic P450 spectral shifts upon substrate binding

  • Circular dichroism to detect conformational changes

  • Fluorescence spectroscopy if intrinsic tryptophan residues are appropriately positioned

How can I design a comprehensive study to investigate CYP82G1 regulation at transcriptional, post-transcriptional, and post-translational levels?

A multilevel analysis of CYP82G1 regulation requires integration of various molecular approaches:

• Transcriptional Regulation:

  • Promoter analysis: Clone the CYP82G1 promoter region and create reporter gene constructs

  • Identify transcription factor binding sites through ChIP-seq or DNA affinity purification

  • Perform yeast one-hybrid assays to identify interacting transcription factors

  • Analyze expression patterns in different tissues and under various stresses

• Post-transcriptional Regulation:

  • RNA stability assays: Treat plants with transcriptional inhibitors and monitor CYP82G1 mRNA decay rates

  • Investigate potential miRNA regulation through bioinformatic prediction and validation

  • Alternative splicing analysis via RT-PCR and RNA-seq

• Post-translational Regulation:

  • Immunoprecipitation followed by LC-MS/MS to identify modifications

  • Site-directed mutagenesis of potential modification sites

  • Protein stability assays in the presence of various signaling molecules or stress conditions

• Protein-Protein Interactions:

  • Co-immunoprecipitation with anti-CYP82G1 antibodies to identify interacting partners

  • Yeast two-hybrid or split-luciferase complementation assays for validation

  • Bimolecular fluorescence complementation (BiFC) for in planta confirmation

What are common challenges in CYP82G1 antibody production and how can they be addressed?

Researchers frequently encounter several challenges when developing CYP82G1 antibodies:

• Low Immunogenicity:

  • Solution: Use carrier proteins (KLH, BSA) conjugated to CYP82G1-specific peptides

  • Select peptides from unique, surface-exposed regions based on structural predictions

  • Consider using multiple peptides to increase chances of success

• Cross-Reactivity with Related P450s:

  • Solution: Conduct thorough blast analyses to identify unique epitopes

  • Use peptides from divergent regions rather than conserved domains

  • Perform extensive validation in knockout mutants and with recombinant proteins

• Poor Antibody Titer:

  • Solution: Optimize immunization protocols with appropriate adjuvants

  • Consider longer immunization schedules with additional booster injections

  • Screen multiple host animals to identify strong responders

• Inconsistent Performance Across Applications:

  • Solution: Develop application-specific antibodies (Western blot vs. immunoprecipitation)

  • Perform affinity purification against the immunizing antigen

  • Establish optimized protocols for each application with appropriate controls

How do I optimize protein extraction protocols for maximum CYP82G1 recovery from plant tissues?

Efficient extraction of membrane-associated proteins like CYP82G1 requires careful optimization:

• Tissue Selection and Preparation:

  • Harvest tissues with high CYP82G1 expression (herbivore-damaged leaves or specific floral tissues)

  • Flash-freeze in liquid nitrogen and grind to a fine powder

  • Maintain cold chain throughout the extraction process

• Buffer Optimization:

  Buffer Component	Recommended Range	Function
  Tris-HCl (pH 7.5-8.0)	50-100 mM	Maintains pH
  NaCl	100-300 mM	Reduces ionic interactions
  Glycerol	10-20%	Stabilizes protein
  EDTA	1-5 mM	Chelates metal ions
  DTT	1-5 mM	Maintains reducing environment
  Detergent (Triton X-100, NP-40)	0.5-1%	Solubilizes membrane proteins
  Protease inhibitor cocktail	As recommended	Prevents degradation

• Extraction Procedure:

  • Test different detergent types and concentrations for optimal membrane protein solubilization

  • Consider sequential extraction with increasing detergent strengths

  • Include ultracentrifugation steps (100,000 × g) to separate membrane fractions

• Verification Methods:

  • Western blotting with anti-CYP82G1 antibody to track protein recovery

  • Activity assays with recovered protein to ensure functional integrity

  • Compare multiple extraction protocols side-by-side to identify optimal conditions

How can CYP82G1 antibodies be applied to study plant-herbivore interaction mechanisms?

CYP82G1 antibodies offer powerful tools for investigating complex plant-herbivore interactions:

• Spatiotemporal Expression Patterns:

  • Immunolocalize CYP82G1 in different tissues before and after herbivore attack

  • Track protein abundance changes in response to different herbivore species

  • Correlate protein levels with TMTT production and herbivore defense efficiency

• Signaling Pathway Elucidation:

  • Investigate how herbivore-induced signals (e.g., jasmonate) affect CYP82G1 protein levels

  • Use co-immunoprecipitation to identify interaction partners in defense signaling cascades

  • Study post-translational modifications that might regulate CYP82G1 activity during herbivory

• Comparative Studies Across Plant Species:

  • If antibody cross-reactivity permits, compare CYP82G1 regulation in different plant species

  • Correlate evolutionary adaptations in CYP82G1 with herbivore pressure

  • Examine potential differential regulation in resistant versus susceptible plant varieties

• Field Applications:

  • Develop immunochromatographic assays for rapid detection of CYP82G1 induction in field settings

  • Monitor protein levels in natural plant populations under varying herbivore pressures

What emerging technologies might enhance our ability to study CYP82G1 function and regulation?

Several cutting-edge approaches have potential to revolutionize CYP82G1 research:

• CRISPR-Based Technologies:

  • Generate precise modifications to study structure-function relationships

  • Create epitope-tagged versions of endogenous CYP82G1 for enhanced detection

  • Develop CYP82G1 reporter lines using knock-in strategies

• Single-Cell Approaches:

  • Single-cell proteomics to detect cell-specific CYP82G1 expression patterns

  • Spatial transcriptomics to correlate CYP82G1 expression with tissue microenvironments

  • Cell-specific metabolomics to link CYP82G1 to TMTT production at cellular resolution

• Cryo-EM and Structural Biology:

  • Determine high-resolution structure of CYP82G1 alone and in complex with substrates

  • Visualize conformational changes during catalytic cycle

  • Enable structure-based design of specific inhibitors or enhancers

• Biosensor Development:

  • Create FRET-based biosensors to monitor CYP82G1 activity in real-time

  • Develop sensors for TMTT production to visualize volatile emission patterns

  • Design synthetic circuits responsive to CYP82G1 activity for engineered plant defense systems