CYP710A4 Antibody

What is CYP710A4 and why is it important in plant research?

CYP710A4 is a cytochrome P450 enzyme belonging to the CYP710A subfamily in plants, particularly well-characterized in Arabidopsis thaliana. It functions as a sterol C-22 desaturase that catalyzes the conversion of β-sitosterol to stigmasterol, which are major sterols in vascular plants .
The importance of CYP710A4 in plant research stems from:

• Its role in sterol metabolism, particularly in modifying the stigmasterol:sitosterol ratio which influences cell membrane properties and stress responses

• Being part of a small cytochrome P450 subfamily with four members (CYP710A1-A4) in Arabidopsis that are evolutionarily related to the yeast sterol C-22 desaturase Erg5p

• Its potential involvement in plant stress responses, as alterations in sterol composition affect membrane properties

• Its significance in understanding plant lipid metabolism pathways

How can I detect CYP710A4 protein expression in plant tissues?

Detection of CYP710A4 protein in plant tissues typically employs antibody-based methods:
Western Blotting Protocol:

• Extract total proteins from plant tissue using appropriate buffer (e.g., Tris-Cl pH 7.2, DTT, NaCl, MgCl₂, EDTA, Triton X-100, and protease inhibitors)

• Quantify protein concentration using Bradford or Amido-black assay

• Separate proteins by SDS-PAGE (typically 10%) and transfer to nitrocellulose membrane

• Block with 5% BSA in TBS

• Incubate with anti-CYP710A4 primary antibody (typically 1:3000-1:5000 dilution)

• Wash with TBS-T and incubate with HRP-conjugated secondary antibody

• Detect using chemiluminescence or colorimetric methods
  Important considerations:

• Include appropriate controls (wild-type and CYP710A4 overexpression samples)

• Normalize loading with housekeeping proteins such as β-actin (42 kDa)

• The expected molecular weight of CYP710A4 is approximately 55 kDa

How specific are antibodies for CYP710A4 compared to other CYP710A family members?

This is an important consideration as Arabidopsis contains four CYP710A family members (CYP710A1-A4) with potential sequence similarity:

• Commercially available or custom-made antibodies against CYP710A4 may exhibit cross-reactivity with other CYP710A family members, particularly CYP710A1, due to sequence homology

• Studies should validate antibody specificity using:

  • Recombinant protein controls of all four CYP710A proteins

  • Protein extracts from knockout and overexpression lines of individual CYP710A genes

  • Peptide competition assays to confirm binding specificity
    For definitive identification of specific isoforms, complementary approaches should be used:

• RT-PCR for mRNA expression analysis of specific CYP710A genes

• Mass spectrometry-based proteomics for unambiguous protein identification

• Functional assays measuring specific enzymatic activities

How should I design experiments to study CYP710A4 expression under different conditions?

Experimental design considerations:

• Control and treatment groups:

  • Wild-type plants as negative controls

  • CYP710A4 overexpression lines as positive controls

  • CYP710A4 knockout/knockdown lines (if available)

  • Various treatment conditions (developmental stages, stress conditions)

• Sample collection strategy:

  • Multiple time points to capture expression dynamics

  • Different tissue types (roots, shoots, leaves, flowers)

  • Biological replicates (minimum of 3) for statistical validity

• Detection methods:

  • Western blotting with anti-CYP710A4 antibodies

  • RT-PCR/qPCR for transcript analysis

  • Enzymatic activity assays for functional validation

• Data analysis:

  • Normalization using housekeeping genes/proteins

  • Densitometric analysis of Western blot bands using software like ImageJ

  • Statistical analysis (ANOVA, t-tests) with appropriate p-value thresholds

What controls should I include when using CYP710A4 antibodies?

Essential controls for CYP710A4 antibody experiments:

• Positive controls:

  • Recombinant CYP710A4 protein (50-100 ng)

  • Protein extracts from CYP710A4 overexpressing plants

• Negative controls:

  • Protein extracts from CYP710A4 knockout mutants (if available)

  • Pre-immune serum control

  • Secondary antibody-only control to detect non-specific binding

• Specificity controls:

  • Peptide competition assay to confirm epitope specificity

  • Recombinant proteins of other CYP710A family members to assess cross-reactivity

• Loading controls:

  • Housekeeping proteins such as β-actin (42 kDa) for Western blot normalization

  • Total protein staining (Ponceau S, Amido Black) for membrane loading verification

• Method controls:

  • Molecular weight markers to confirm protein size (~55 kDa for CYP710A4)

How can I optimize protein extraction for maximum CYP710A4 detection?

CYP710A4 is a membrane-associated cytochrome P450 enzyme, requiring specific extraction strategies:
Optimized extraction protocol:

• Buffer composition:

  • Base buffer: 10-20 mM Tris-Cl (pH 7.2-8.0)

  • Reducing agent: 5 mM DTT

  • Salt: 10 mM NaCl, 1.5 mM MgCl₂

  • Metal chelator: 0.1-0.5 mM EDTA

  • Detergent: 0.4-0.5% Triton X-100 (critical for membrane protein solubilization)

  • Protease inhibitors: 0.3 mM PMSF and protease inhibitor cocktail

• Extraction procedure:

  • Flash-freeze tissue in liquid nitrogen

  • Grind to fine powder while frozen

  • Add extraction buffer (1:3 w/v ratio)

  • Use multiple freeze-thaw cycles to improve membrane disruption

  • Sonicate samples on ice (3-5 short pulses)

  • Centrifuge at 10,000-100,000 g to separate soluble fraction

• Post-extraction handling:

  • Keep samples cold throughout the procedure

  • Add glycerol (10%) for protein stability

  • Avoid repeated freeze-thaw cycles of extracts

  • Use freshly prepared samples when possible

How can CYP710A4 antibodies be used to investigate protein-protein interactions in sterol biosynthesis pathways?

Methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use anti-CYP710A4 antibodies to pull down CYP710A4 and associated proteins

  • Identify interacting partners by mass spectrometry

  • Validate interactions with reverse Co-IP using antibodies against putative partners

  • Controls should include non-specific IgG and lysates from CYP710A4 knockout plants

• Proximity labeling approaches:

  • Generate fusion proteins of CYP710A4 with BioID or APEX2

  • Express in plants to label proximal proteins

  • Identify labeled proteins by streptavidin pulldown and mass spectrometry

  • Validate with CYP710A4 antibodies in co-localization studies

• Bimolecular Fluorescence Complementation (BiFC):

  • Create fusion constructs of CYP710A4 with split fluorescent protein fragments

  • Co-express with candidate interactors in plant cells

  • Verify protein expression levels by Western blot with CYP710A4 antibodies

  • Visualize interactions by fluorescence microscopy

• Analysis considerations:

  • Focus on known sterol biosynthesis enzymes as potential interactors

  • Consider interactions with NADPH-P450 reductase, which is essential for P450 function

  • Investigate potential regulatory proteins (kinases, phosphatases)

What are the best methods to analyze subcellular localization of CYP710A4 using antibodies?

Subcellular localization approaches:

• Immunofluorescence microscopy:

  • Fix plant tissues with paraformaldehyde

  • Permeabilize with detergent

  • Block with BSA or normal serum

  • Incubate with anti-CYP710A4 primary antibody

  • Detect with fluorophore-conjugated secondary antibody

  • Co-stain with organelle markers (ER, Golgi, plasma membrane)

  • Image using confocal microscopy

• Subcellular fractionation with immunoblotting:

  • Isolate different cellular fractions (microsomal, cytosolic, etc.)

  • Perform Western blotting with anti-CYP710A4 antibodies

  • Include marker proteins for different compartments (e.g., BiP for ER, PMA for plasma membrane)

  • Quantify relative distribution across fractions

• Electron microscopy immunogold labeling:

  • Fix tissue samples in glutaraldehyde/paraformaldehyde

  • Embed in resin and section

  • Incubate with anti-CYP710A4 antibody

  • Detect with gold-conjugated secondary antibody

  • Image using transmission electron microscope

  • Quantify gold particle distribution across cellular compartments
    Expected localization:

• As a cytochrome P450, CYP710A4 is likely anchored in the endoplasmic reticulum membrane

• The catalytic domain faces the cytosolic side of the ER membrane

How can I analyze changes in CYP710A4 protein levels in relation to sterol composition?

Integrated analytical approach:

• Protein quantification:

  • Western blot with anti-CYP710A4 antibodies on plant samples

  • Include recombinant CYP710A4 protein standards for absolute quantification

  • Normalize to loading controls and perform densitometric analysis

• Sterol extraction and analysis:

  • Extract sterols using ethyl acetate or similar solvents

  • Derivatize with BSTFA for GC-MS analysis

  • Quantify β-sitosterol and stigmasterol levels

  • Calculate stigmasterol:sitosterol ratios

• Experimental design:

  • Compare wild-type, CYP710A4 knockdown, and CYP710A4 overexpressing plants

  • Include time-course experiments following induction of CYP710A4 expression

  • Analyze different tissues and developmental stages

• Correlation analysis:

  • Plot CYP710A4 protein levels against stigmasterol:sitosterol ratios

  • Perform regression analysis to determine relationship strength

  • Consider time-lag effects between protein expression and metabolite changes

• Validation experiments:

  • In vitro enzymatic assays using recombinant CYP710A4 with β-sitosterol substrate

  • Measure reaction kinetics (Km, kcat) to correlate with in vivo observations

What are common problems when using CYP710A4 antibodies and how can they be resolved?

Common challenges and solutions:

• Weak or no signal:

  • Cause: Low antibody concentration, protein degradation, insufficient antigen

  • Solution: Increase antibody concentration, optimize protein extraction with fresh protease inhibitors, increase protein loading amount, enhance detection system sensitivity

• Multiple bands or non-specific binding:

  • Cause: Cross-reactivity with other CYP710A family members, degradation products

  • Solution: Use more stringent washing conditions, optimize blocking (try 5% BSA instead of milk), perform peptide competition assays, use knockout mutants as negative controls

• High background signal:

  • Cause: Insufficient blocking, excessive antibody concentration

  • Solution: Increase blocking time/concentration, dilute antibody further, add 0.1-0.3% Tween-20 in washing steps, pre-absorb antibody with plant extract from CYP710A4 knockout

• Inconsistent results:

  • Cause: Variable extraction efficiency, protein modification differences

  • Solution: Standardize extraction protocol, use internal controls, increase biological replicates

• Variable detection between experiments:

  • Cause: Antibody lot variability, protein modification differences

  • Solution: Use the same antibody lot for comparative studies, include standard samples across experiments for normalization

How can I quantitatively analyze CYP710A4 expression data from Western blots?

Quantitative analysis workflow:

• Image acquisition:

  • Capture images within the linear range of detection

  • Use a calibrated imaging system (ChemiDoc, LI-COR Odyssey)

  • Include a standard curve of recombinant CYP710A4 (50-100 ng)

• Densitometric analysis:

  • Use software like ImageJ to quantify band intensities

  • Define consistent measurement area for all bands

  • Subtract background signal from an adjacent area

• Normalization approaches:

  • Normalize to housekeeping protein (β-actin)

  • Alternatively, normalize to total protein (Ponceau S staining)

  • For absolute quantification, compare to recombinant protein standard curve

• Statistical analysis:

  • Perform experiments with at least three biological replicates

  • Apply appropriate statistical tests (t-test, ANOVA)

  • Report data as mean ± standard deviation with p-values

  • Use significance thresholds: * p < 0.05; ** p < 0.01; *** p < 0.005

• Data visualization:

  • Present normalized data in bar graphs with error bars

  • Include representative Western blot images

  • Show fold-change relative to control conditions

How should I interpret unexpected changes in CYP710A4 expression patterns?

Interpretation framework for unexpected results:

• Biological considerations:

  • Post-translational modifications might affect antibody recognition

  • Protein stability could be altered under experimental conditions

  • Alternative splicing might produce variant isoforms

  • Protein localization changes might affect extraction efficiency

• Analytical verification steps:

  • Confirm results with multiple detection methods (Western blot, RT-PCR, enzyme activity)

  • Verify with different antibodies targeting different epitopes of CYP710A4

  • Check for changes in other CYP710A family members

• Experimental design validation:

  • Rule out technical artifacts through appropriate controls

  • Increase biological and technical replicates

  • Test alternative protein extraction methods

  • Consider tissue-specific or cell-type-specific expression patterns

• Metabolite correlation:

  • Analyze whether unexpected protein changes correlate with altered sterol profiles

  • Measure enzyme activity to determine if protein changes reflect functional changes

  • Investigate potential feedback mechanisms in sterol biosynthesis pathway

How can CYP710A4 antibodies be used in comparative studies across plant species?

Cross-species research approaches:

• Antibody selection strategies:

  • Design antibodies against conserved epitopes across plant CYP710A proteins

  • Test antibody cross-reactivity with recombinant proteins from different species

  • Consider using multiple antibodies targeting different regions of the protein

• Experimental design for comparative studies:

  • Include phylogenetically diverse plant species (monocots, dicots)

  • Sample equivalent tissues at comparable developmental stages

  • Optimize protein extraction protocols for each species

  • Use internal loading controls appropriate for each species

• Data analysis considerations:

  • Normalize expression to appropriate reference proteins for each species

  • Consider evolutionary relationships when interpreting expression patterns

  • Correlate with sterol profiles across species

  • Analyze sequence conservation in relation to antibody recognition sites

• Applications in evolutionary biology:

  • Study CYP710A4 expression across land plant evolution

  • Investigate functional conservation of sterol C-22 desaturases

  • Correlate enzyme expression with adaptation to different environments

What is the relationship between CYP710A4 and other sterol biosynthesis enzymes in plant stress responses?

Integrated analysis approaches:

• Multi-protein expression analysis:

  • Develop antibody panels against key sterol biosynthesis enzymes

  • Perform Western blot analysis of multiple enzymes from the same samples

  • Create protein expression profiles across stress conditions and time points

• Correlation with sterol metabolites:

  • Monitor changes in sterol composition (sitosterol, stigmasterol) under stress

  • Calculate enzyme:metabolite ratios to identify rate-limiting steps

  • Analyze esterified versus free sterols to assess metabolic flux

• Functional interaction studies:

  • Use CYP710A4 antibodies in co-immunoprecipitation experiments

  • Identify stress-specific protein interactions

  • Investigate post-translational modifications affecting enzyme activity

• Stress-specific considerations:

  • Focus on stresses known to affect membrane properties

  • Consider both abiotic stresses (temperature, drought) and biotic stresses (pathogens)

  • Investigate tissue-specific responses

• Regulatory network analysis:

  • Identify transcription factors controlling CYP710A4 expression under stress

  • Study correlation between protein and mRNA levels during stress responses

  • Analyze promoter elements for stress-responsive motifs

How can CYP710A4 antibodies be used in studies of membrane microdomain composition?

Methodological approaches:

• Membrane fractionation:

  • Isolate detergent-resistant membrane fractions (lipid rafts)

  • Perform Western blot analysis with CYP710A4 antibodies

  • Compare distribution in different membrane fractions

  • Correlate with sterol composition of membrane fractions

• Immunolocalization in membrane domains:

  • Use super-resolution microscopy (STORM, PALM) with CYP710A4 antibodies

  • Co-localize with membrane domain markers

  • Analyze spatial distribution patterns

• Experimental manipulations:

  • Compare wild-type plants with CYP710A4 overexpressors and knockdowns

  • Analyze changes in membrane protein distribution

  • Correlate with alterations in sterol composition and membrane fluidity

• Applications in cellular biology:

  • Study the role of stigmasterol in organizing membrane microdomains

  • Investigate protein sorting mechanisms in the endomembrane system

  • Analyze signal transduction complexes in specialized membrane domains

Table 1: Comparative Properties of CYP710A Family Members in Arabidopsis

Table 2: Recommended Antibody Dilutions and Applications for CYP710A4 Detection

Table 3: Effect of CYP710A4 Overexpression on Sterol Composition in Arabidopsis