CYP97B3 Antibody

What is CYP97B3 and what role does it play in plants?

CYP97B3 (Cytochrome P450 97B3) is a member of the cytochrome P450 superfamily encoded by the gene At1g31800 in Arabidopsis thaliana. It functions as part of the carotenoid biosynthesis pathway in plants. Unlike CYP97A3 (LUT5) and CYP97C1 (LUT1) which have been well-characterized for their roles in carotenoid hydroxylation, CYP97B3's precise function in the carotenoid pathway is less well-defined. Research suggests it may be involved in secondary modifications of carotenoid structures or in parallel biochemical pathways affecting carotenoid accumulation .

What are the recommended methods for using CYP97B3 antibodies in Western blot analysis?

For optimal Western blot detection of CYP97B3:

• Extraction: Use a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail for total protein extraction from plant tissues.

• Sample preparation: Due to CYP97B3's membrane association, avoid boiling samples to prevent aggregation. Instead, incubate at 37°C for 30 minutes in sample buffer.

• Gel separation: Use 10% SDS-PAGE for effective resolution of CYP97B3 (≈58 kDa).

• Transfer: PVDF membranes typically work better than nitrocellulose for hydrophobic proteins like CYP97B3.

• Blocking: 5% non-fat dry milk in TBST for 2 hours at room temperature.

• Primary antibody: Dilute CYP97B3 antibody (typically 1:1000 to 1:2000) in blocking buffer and incubate overnight at 4°C.

• Detection: Use appropriate secondary antibody and visualization system.

Include positive controls and verify results with Arabidopsis cyp97b3 mutant extracts as negative controls.

How can CYP97B3 antibodies be used to study protein localization in plant cells?

For immunolocalization of CYP97B3 in plant tissues:

• Tissue fixation: Fix plant tissues in 4% paraformaldehyde in PBS (pH 7.4) for 2-4 hours.

• Embedding and sectioning: Embed fixed tissues in paraffin or resin and create 5-10 μm sections.

• Antigen retrieval: Treat sections with citrate buffer (pH 6.0) at 95°C for 10-15 minutes.

• Blocking: Use 3% BSA in PBS with 0.1% Triton X-100 for 1 hour.

• Primary antibody: Apply diluted CYP97B3 antibody (1:100 to 1:500) and incubate overnight at 4°C.

• Secondary antibody: Use fluorophore-conjugated secondary antibody appropriate for your detection system.

• Counterstaining: DAPI for nuclei visualization.

• Imaging: Confocal microscopy for high-resolution localization.

This approach can help determine the subcellular localization of CYP97B3, which is expected to associate with the endoplasmic reticulum membrane like other plant P450 enzymes.

Can CYP97B3 antibodies be used for immunoprecipitation to study protein interactions?

Yes, CYP97B3 antibodies can be used for immunoprecipitation (IP) studies to identify protein interaction partners. The protocol should be optimized as follows:

• Protein extraction: Use a mild detergent buffer (0.5-1% NP-40 or digitonin) to preserve protein-protein interactions.

• Pre-clearing: Incubate plant lysate with protein A/G beads to reduce non-specific binding.

• Immunoprecipitation: Add CYP97B3 antibody (2-5 μg) to pre-cleared lysate and incubate overnight at 4°C with gentle rotation.

• Capture: Add protein A/G beads for 2-4 hours at 4°C.

• Washing: Use progressively stringent wash buffers to remove non-specific interactions.

• Elution and analysis: Elute bound proteins and analyze by Western blot or mass spectrometry.

This approach can help identify proteins that interact with CYP97B3 in the carotenoid biosynthesis pathway or reveal novel functional connections .

Why might CYP97B3 antibody yield weak signals in Western blot analysis?

Several factors can contribute to weak CYP97B3 detection signals:

• Low protein expression: CYP97B3 may be expressed at low levels under standard growth conditions. Consider using stress conditions (high light, drought) that might upregulate carotenoid biosynthesis genes.

• Protein degradation: Ensure fresh protease inhibitors are included in all extraction buffers.

• Inefficient extraction: CYP97B3 is a membrane-associated protein; standard aqueous extraction may be insufficient. Include detergents like 1% Triton X-100 or 0.5% sodium deoxycholate in extraction buffers.

• Inefficient transfer: Membrane proteins can be difficult to transfer efficiently. Consider longer transfer times or semi-dry transfer systems.

• Antibody dilution: Test different antibody concentrations (1:500 to 1:5000) to optimize signal-to-noise ratio.

• Detection system sensitivity: Switch to more sensitive chemiluminescent substrates or consider fluorescent-based detection systems.

Comparing results with appropriate positive controls can help identify the specific issue in your experimental workflow.

How can cross-reactivity issues with CYP97B3 antibodies be addressed?

To minimize cross-reactivity concerns:

• Epitope analysis: Review the epitope used for antibody generation and compare its sequence to other plant P450 enzymes, particularly CYP97A3 and CYP97C1.

• Pre-absorption: Incubate the antibody with recombinant proteins or peptides from potentially cross-reactive P450 enzymes before use.

• Knockout validation: Always include protein extracts from cyp97b3 knockout mutants as negative controls.

• Western blot optimization: Increase washing stringency and optimize blocking conditions to reduce non-specific binding.

• Competitive ELISA: Perform competitive ELISA assays with related peptides to quantify cross-reactivity.

• Alternative detection: Consider using alternative detection methods like mass spectrometry for critical experiments requiring absolute specificity.

Implementing these approaches ensures more reliable and interpretable experimental results.

What are the recommended storage conditions to maintain CYP97B3 antibody activity?

To preserve antibody functionality:

• Storage temperature: Store antibody aliquots at -20°C for long-term storage; avoid repeated freeze-thaw cycles.

• Working dilutions: Store at 4°C with preservatives (0.02% sodium azide) for up to 2 weeks.

• Aliquoting: Divide stock antibody into single-use aliquots to prevent degradation from repeated freeze-thaw cycles.

• Preservatives: Include carrier proteins (0.1-1% BSA) and preservatives in storage buffer.

• Stability testing: Periodically test antibody activity against a standard sample to monitor degradation over time.

• Avoid contamination: Use sterile technique when handling antibodies.

Following these guidelines will maximize antibody shelf-life and experimental reproducibility .

How can researchers quantify CYP97B3 protein expression levels using antibody-based techniques?

For reliable quantification of CYP97B3:

• Western blot quantification:

  • Use a standard curve with recombinant CYP97B3 protein at known concentrations

  • Employ housekeeping proteins (actin, tubulin) as loading controls

  • Use digital imaging and analysis software for densitometry measurements

• ELISA quantification:

  • Develop a sandwich ELISA using CYP97B3 antibody as capture or detection antibody

  • Generate a standard curve using purified recombinant CYP97B3

  • Calculate sample concentrations based on the standard curve

• Flow cytometry (for protoplasts):

  • Label fixed and permeabilized protoplasts with fluorophore-conjugated CYP97B3 antibody

  • Use flow cytometry to measure fluorescence intensity

  • Compare with appropriate negative and positive controls

These approaches provide complementary data for robust quantification of CYP97B3 protein levels under different experimental conditions .

How should researchers interpret changes in CYP97B3 protein levels in response to environmental stresses?

When analyzing CYP97B3 expression changes under stress conditions:

• Temporal considerations: Examine multiple time points as transient versus sustained changes may indicate different regulatory mechanisms.

• Cross-pathway analysis: Correlate CYP97B3 expression with other carotenoid biosynthesis genes (PSY, PDS, Z-ISO, ZDS, CRTISO, εLCY, βLCY, CYP97A3, CYP97C1) to understand pathway-level responses .

• Physiological context: Connect CYP97B3 changes to measurable changes in carotenoid profiles using HPLC or LC-MS/MS analysis.

• Subcellular redistribution: Consider that total protein levels may remain constant while subcellular localization changes.

• Post-translational modifications: Assess potential changes in CYP97B3 phosphorylation or other modifications using phospho-specific antibodies or mass spectrometry.

• Mutant comparisons: Compare stress responses in wild-type plants versus cyp97b3 mutants to determine functional significance.

This comprehensive approach provides deeper insights into CYP97B3's role in stress adaptation mechanisms.

How can researchers use CYP97B3 antibodies in chromatin immunoprecipitation (ChIP) studies?

While CYP97B3 itself is not a transcription factor, its antibodies might be useful in ChIP studies if investigating:

• Potential chromatin association: Under specific conditions, some metabolic enzymes can associate with chromatin. If preliminary evidence suggests this for CYP97B3, ChIP protocol would involve:

  • Crosslinking plant tissue with formaldehyde

  • Sonicating chromatin to appropriate fragment size (200-500 bp)

  • Immunoprecipitating with CYP97B3 antibody

  • Analyzing associated DNA by qPCR or sequencing

• Transcription factor interactions: If studying transcription factors that regulate CYP97B3, a reverse approach would involve:

  • ChIP with antibodies against candidate transcription factors

  • qPCR analysis of enrichment at the CYP97B3 promoter region

Such studies could reveal novel regulatory mechanisms for carotenoid biosynthesis genes.

What approaches can be used to study post-translational modifications of CYP97B3 using antibodies?

To investigate post-translational modifications (PTMs) of CYP97B3:

• Phosphorylation analysis:

  • Immunoprecipitate CYP97B3 using specific antibodies

  • Analyze by Western blot with phospho-specific antibodies

  • Perform mass spectrometry analysis of immunoprecipitated protein to identify phosphorylation sites

• Ubiquitination detection:

  • Co-immunoprecipitation with anti-ubiquitin and anti-CYP97B3 antibodies

  • Use deubiquitinating enzyme inhibitors during extraction

  • Analyze high molecular weight bands by mass spectrometry

• Other PTMs (glycosylation, acetylation):

  • Combine CYP97B3 immunoprecipitation with specific PTM detection methods

  • Use PTM-specific enzymes (glycosidases, deacetylases) to confirm modifications

Understanding CYP97B3 PTMs could reveal regulatory mechanisms controlling enzyme activity, stability, and subcellular localization.

How can CYP97B3 antibodies be used in combination with CRISPR-Cas9 gene editing to study protein function?

Integration of CYP97B3 antibodies with CRISPR-Cas9 technology enables sophisticated functional studies:

• Validation of knockout efficiency:

  • Use CYP97B3 antibodies to confirm complete protein absence in CRISPR-generated knockout lines

  • Quantify residual protein in partial knockouts or alternative splice variants

• Epitope tagging verification:

  • When introducing epitope tags (HA, FLAG, GFP) to CYP97B3 via CRISPR, use native CYP97B3 antibodies to confirm normal expression levels compared to untagged protein

• Domain function analysis:

  • Generate domain-specific deletions via CRISPR

  • Use CYP97B3 antibodies to assess expression, stability, and localization of truncated proteins

• Proximity labeling studies:

  • Create CRISPR knock-ins fusing CYP97B3 with BioID or APEX2

  • Use CYP97B3 antibodies to confirm fusion protein expression

  • Identify proximal proteins by streptavidin pulldown and mass spectrometry

This integrated approach provides comprehensive insights into CYP97B3 function within cellular contexts .

How does CYP97B3 antibody detection compare between different plant species?

When using CYP97B3 antibodies across plant species:

• Sequence homology: Evaluate sequence conservation of the antibody epitope across target species. Higher conservation (>80%) suggests greater likelihood of cross-reactivity.

• Species validation: Test antibody reactivity in each new species using:

  • Western blot analysis of total protein extracts

  • Immunoprecipitation followed by mass spectrometry confirmation

  • Preabsorption controls with recombinant proteins

• Optimization requirements:

  Plant Species	Expected Cross-Reactivity	Recommended Dilution	Special Considerations
  Arabidopsis thaliana	High (original target)	1:1000-1:2000	Standard protocols
  Brassica species	Moderate to High	1:500-1:1000	Increased antibody concentration
  Tomato/Potato	Low to Moderate	1:250-1:500	Extended incubation times
  Monocots (rice, wheat)	Very Low	1:100-1:250	May require alternative extraction methods

• Negative controls: Include appropriate negative controls (preferably gene knockout lines) from each species being tested.

This systematic approach ensures reliable cross-species applications of CYP97B3 antibodies.

What are the differences between studying CYP97B3 using antibody-based approaches versus transcriptional analysis?

Understanding the complementary nature of protein and transcript analyses:

• Information content comparison:

  Parameter	Antibody-Based Detection	Transcript Analysis
  Post-transcriptional regulation	Detectable	Not captured
  Protein stability/turnover	Measurable	Not detected
  Subcellular localization	Can be determined	Cannot be determined
  Protein modifications	Potentially detectable	Not detectable
  High-throughput capability	Limited	Extensive (RNA-seq)
  Quantitative accuracy	Moderate (Western blot) to High (ELISA)	High (qRT-PCR, RNA-seq)

• Integrated analysis approach:

  • Correlate protein levels (antibody detection) with mRNA levels (qRT-PCR)

  • Investigate discrepancies to identify post-transcriptional regulation

  • Use transcriptomics to identify conditions for further protein-level studies

  • Employ ribosome profiling alongside antibody detection to assess translational efficiency

• Experimental design considerations:

  • Temporal dynamics: Transcript changes typically precede protein changes

  • Sample requirements: Antibody methods often require more biological material

  • Specificity: Transcript detection can achieve higher specificity through primer design

This comparative understanding helps researchers select appropriate approaches based on specific research questions .

How might advanced imaging techniques enhance CYP97B3 antibody applications?

Emerging imaging technologies open new possibilities for CYP97B3 research:

• Super-resolution microscopy:

  • STED or STORM microscopy can resolve CYP97B3 localization beyond the diffraction limit

  • Potential to visualize CYP97B3 organization within membrane microdomains

  • Co-localization studies with other carotenoid biosynthetic enzymes at nanoscale resolution

• Live-cell imaging:

  • Combine antibody fragment technologies (nanobodies) against CYP97B3 with cell-penetrating peptides

  • Monitor dynamic changes in CYP97B3 localization in response to stimuli

  • Develop novel visualization tools for studying membrane-associated enzymes in living plant cells

• Correlative light and electron microscopy (CLEM):

  • Use CYP97B3 antibodies for immunogold labeling

  • Combine with fluorescence microscopy for multiscale imaging

  • Achieve molecular resolution of CYP97B3 in its cellular context

These advanced techniques could reveal unprecedented details about CYP97B3 organization and dynamics in plant cells .

What role might CYP97B3 antibodies play in understanding plant stress response mechanisms?

CYP97B3 antibodies can provide valuable insights into stress-related carotenoid metabolism:

• Abiotic stress studies:

  • Monitor CYP97B3 protein levels during high light, drought, or temperature stress

  • Correlate with changes in specific carotenoid profiles using HPLC analysis

  • Determine if CYP97B3 is rate-limiting under specific stress conditions

• Oxidative stress response:

  • Investigate potential roles of CYP97B3 in producing carotenoid derivatives that function as signaling molecules

  • Use antibodies to track protein abundance during oxidative stress

  • Combine with metabolomic profiling to identify novel carotenoid-derived compounds

• Stress signaling integration:

  • Study co-localization with stress signaling components

  • Determine if CYP97B3 undergoes stress-induced relocalization

  • Investigate potential protein-protein interactions that change under stress conditions

This research direction could reveal novel functions of CYP97B3 beyond its catalytic role in carotenoid biosynthesis .

How can researchers develop an ELISA-based detection system for CYP97B3 quantification?

Developing a reliable ELISA for CYP97B3 requires:

• Assay design considerations:

  • Sandwich ELISA: Requires two antibodies recognizing different epitopes (capture and detection)

  • Competitive ELISA: Better for small proteins or when only one antibody is available

  • Direct ELISA: Simplest approach but may have higher background

• Development protocol:

  • Express and purify recombinant CYP97B3 protein for standards

  • Optimize antibody concentrations (typically 1-10 μg/ml for coating)

  • Determine optimal blocking conditions (typically 1-5% BSA or non-fat milk)

  • Establish sample preparation protocol (detergent solubilization critical for membrane proteins)

  • Generate standard curve with recombinant CYP97B3 (typical range: 0.1-100 ng/ml)

• Validation requirements:

  • Demonstrate specificity using cyp97b3 knockout plant extracts

  • Assess matrix effects using spike-and-recovery experiments

  • Determine assay precision (intra- and inter-assay variability <15%)

  • Establish lower limit of quantification (typically 0.1-1 ng/ml)

This quantitative tool would enable high-throughput analysis of CYP97B3 expression across multiple samples and conditions .

What approaches can be used to study the interaction between CYP97B3 and other carotenoid biosynthetic enzymes?

Investigating enzyme-enzyme interactions within the carotenoid pathway:

• Co-immunoprecipitation strategies:

  • Immunoprecipitate CYP97B3 using specific antibodies

  • Analyze co-precipitated proteins by Western blot or mass spectrometry

  • Confirm interactions with reciprocal co-IP experiments

• Proximity labeling approaches:

  • Generate transgenic plants expressing CYP97B3 fused to BioID or APEX2

  • Activate proximity labeling and purify biotinylated proteins

  • Identify labeled proteins using mass spectrometry

  • Validate potential interactions using CYP97B3 antibodies

• Fluorescence-based interaction assays:

  • Förster resonance energy transfer (FRET) between fluorescently-tagged proteins

  • Bimolecular fluorescence complementation (BiFC) in plant protoplasts

  • Use CYP97B3 antibodies to confirm expression levels of fusion proteins

• In vitro reconstitution:

  • Express and purify recombinant carotenoid biosynthetic enzymes

  • Perform pull-down assays with CYP97B3 antibodies

  • Analyze enzyme complex formation using size exclusion chromatography

These approaches could reveal important protein-protein interactions that regulate carotenoid biosynthesis pathway flux and efficiency .

What controls should be included when using CYP97B3 antibodies for immunoblotting experiments?

Comprehensive controls ensure reliable interpretation of CYP97B3 immunoblot results:

• Essential controls:

  Control Type	Description	Purpose
  Positive control	Recombinant CYP97B3 protein	Confirms antibody functionality
  Negative control	Extract from cyp97b3 knockout	Verifies specificity
  Loading control	Housekeeping protein (actin, tubulin)	Normalizes for protein loading
  Secondary antibody-only	Omit primary antibody	Identifies non-specific secondary binding
  Pre-immune serum	For polyclonal antibodies	Establishes baseline reactivity

• Method validation controls:

  • Peptide competition: Pre-incubate antibody with immunizing peptide

  • Cross-species validation: Test antibody against purified orthologous proteins

  • Denaturation sensitivity: Compare native vs. denatured sample detection

• Technical considerations:

  • Molecular weight markers: CYP97B3 should appear at approximately 58 kDa

  • Extraction buffer optimization: Compare different detergents and solubilization methods

  • Transfer efficiency verification: Use reversible total protein staining (Ponceau S)

These controls provide a framework for rigorous validation of CYP97B3 antibody specificity and reliability .

How can researchers assess antibody lot-to-lot variability in CYP97B3 detection?

Managing antibody consistency across different production lots:

• Quantitative comparison protocol:

  • Prepare a standard reference sample (plant extract or recombinant protein)

  • Aliquot and store at -80°C for long-term use

  • Test each new antibody lot against this standard

  • Compare signal intensity, background, and specificity

• Performance metrics to evaluate:

  • Detection sensitivity: Minimum detectable amount of target protein

  • Signal-to-noise ratio: Specific signal versus background

  • Epitope recognition: Peptide competition assay results

  • Cross-reactivity profile: Testing against related proteins

• Documentation and standardization:

  • Maintain detailed records of antibody performance

  • Establish acceptance criteria for new lots

  • Consider creating internal reference standards

  • Implement quality control testing before using new lots in critical experiments

This systematic approach minimizes variability and ensures experimental reproducibility across studies using different antibody lots .

How might single-cell proteomics approaches utilize CYP97B3 antibodies?

Emerging single-cell technologies create new possibilities for CYP97B3 research:

• Mass cytometry (CyTOF) applications:

  • Conjugate CYP97B3 antibodies with rare earth metal isotopes

  • Analyze protein expression at single-cell resolution in plant protoplasts

  • Simultaneously detect multiple proteins in carotenoid biosynthesis pathway

  • Correlate CYP97B3 expression with cell type-specific markers

• Microfluidic antibody-based detection:

  • Capture individual protoplasts in microfluidic chambers

  • Perform in situ immunodetection of CYP97B3

  • Combine with single-cell transcriptomics for multi-omics analysis

  • Investigate cell-to-cell variability in CYP97B3 expression

• Technical considerations:

  • Antibody validation at single-cell level critical (signal specificity)

  • Protocol optimization for plant cell wall digestion and protoplast preparation

  • Development of appropriate normalization strategies

  • Integration with spatial information when possible

These emerging technologies could reveal previously undetectable cell-specific roles of CYP97B3 in plant development and stress responses .

What research questions about CYP97B3 remain unanswered that antibody-based approaches could help address?

Critical knowledge gaps that could be addressed using CYP97B3 antibodies:

• Regulatory mechanisms:

  • Does CYP97B3 undergo tissue-specific or developmental post-translational modifications?

  • Are there protein-protein interactions that regulate CYP97B3 activity?

  • Does CYP97B3 form complexes with other carotenoid biosynthetic enzymes?

• Subcellular dynamics:

  • Does CYP97B3 relocalize in response to environmental stimuli?

  • Is CYP97B3 present in specific membrane microdomains?

  • Are there specialized plastid subcompartments where CYP97B3 is concentrated?

• Metabolic channeling:

  • Does CYP97B3 participate in metabolon formation?

  • How does the spatial organization of CYP97B3 relative to other pathway enzymes affect pathway flux?

  • Are there direct substrate transfers between sequential enzymes?

• Non-canonical functions:

  • Does CYP97B3 have moonlighting functions beyond carotenoid biosynthesis?

  • Could CYP97B3 be involved in stress signaling pathways?

  • Are there tissue-specific isoforms or splice variants with distinct functions?

Addressing these questions using CYP97B3 antibodies could significantly advance our understanding of carotenoid biosynthesis regulation and function in plants .