CYP71B13 Antibody

What is CYP71B13 and what role does it play in plant biology?

CYP71B13 is a cytochrome P450 enzyme that likely functions in plant secondary metabolism and defense responses, similar to other CYP71 family members like CYP71A13 and CYP71B15. These enzymes are often involved in the biosynthesis of phytoalexins, which are antimicrobial compounds produced by plants in response to pathogen infection. CYP71A13, for example, is involved in the biosynthesis of camalexin, a major phytoalexin in Arabidopsis that is synthesized in response to stress factors such as pathogen infection, UV light exposure, and heavy metal treatment . By extrapolation, CYP71B13 may play a similar role in plant defense mechanisms, though its exact function would need to be confirmed through targeted studies.

How do CYP71B13 antibodies differ from other cytochrome P450 antibodies?

CYP71B13 antibodies are specifically designed to recognize and bind to CYP71B13 proteins, distinguishing them from other cytochrome P450 enzymes. This specificity is crucial for accurate detection and analysis in research applications. While cytochrome P450 enzymes share structural similarities, each antibody must be validated for specificity to ensure it does not cross-react with other closely related enzymes like CYP71A12, CYP71A13, or CYP71B15. For example, research on CYP71B15 utilized specific antibodies that could distinguish it from other P450 enzymes during co-immunoprecipitation experiments . Similar validation would be necessary for CYP71B13 antibodies to ensure they do not cross-react with these related enzymes.

What are the primary applications of CYP71B13 antibodies in plant research?

CYP71B13 antibodies can be utilized for various research applications including:

• Western blotting to detect and quantify CYP71B13 protein expression

• Immunohistochemistry to localize CYP71B13 within plant tissues

• Co-immunoprecipitation (co-IP) to identify protein-protein interactions

• Flow cytometry to analyze CYP71B13 expression in different cell populations

• Functional studies to understand the role of CYP71B13 in plant metabolism and defense
  Similar to studies with CYP71B15, CYP71B13 antibodies could be used to investigate potential roles in metabolic complexes or "metabolons" that facilitate efficient biosynthesis of plant defense compounds .

What are the recommended methods for validating CYP71B13 antibody specificity?

When validating CYP71B13 antibody specificity, researchers should consider multiple approaches:

• Western blotting with recombinant protein: Express and purify recombinant CYP71B13 with appropriate tags (e.g., His-tag) and confirm antibody recognition by Western blot. Similar approaches were used to validate antibody specificity in CD73 research where "the extracellular domain of CD73 with a C-terminal His-tag as a recombinant protein" was constructed and purified, followed by Western blotting to confirm antibody specificity .

• Comparison with knockout/knockdown controls: Use plant materials with knocked-out or knocked-down CYP71B13 expression as negative controls to confirm antibody specificity.

• Cross-reactivity testing: Test the antibody against related cytochrome P450 enzymes, particularly those within the CYP71 family (CYP71A12, CYP71A13, CYP71B15) to ensure specificity.

• Mass spectrometry validation: Following immunoprecipitation with the CYP71B13 antibody, analyze the pulled-down proteins by mass spectrometry to confirm the presence of CYP71B13 and assess any cross-reactivity with other proteins. This approach was used effectively in research with other antibodies, as seen in the CD73 antibody study where "mass spectrometry revealed 5′-nucleotidase (UniProt:P21589) and heat shock 70 kDa protein 1A (UniProt:P0DMV8) and 1B (UniProt:P0DMV9) as candidates" .

What is the optimal sample preparation protocol for detecting CYP71B13 in plant tissues?

For optimal detection of CYP71B13 in plant tissues, consider the following protocol:

• Tissue selection: Choose tissues and conditions where CYP71B13 expression is expected to be highest, potentially in response to pathogen infection or other stress conditions, similar to CYP71B15 which "was only observed in cells in close proximity to successful pathogen infection" .

• Sample preparation:

  • Grind plant tissue in liquid nitrogen to a fine powder

  • Extract proteins using a buffer suitable for membrane proteins (as CYP71B13 is likely membrane-bound like other P450 enzymes)

  • Include protease inhibitors to prevent protein degradation

  • Consider microsomal fractionation to enrich for ER-localized proteins, as cytochrome P450 enzymes typically localize to the ER membrane

• Protein solubilization: Use mild detergents such as Triton X-100 or CHAPS to solubilize membrane proteins without denaturing them, especially for applications like co-IP or immunohistochemistry.

• Induction treatments: Consider treating plants with pathogens, UV irradiation, or chemical elicitors to induce CYP71B13 expression prior to sample collection, as was done for CYP71B15 where "strong GFP signal was observed in response to Botrytis cinerea infection, whereas the signal was absent in untreated leaves" .

How should researchers troubleshoot weak or non-specific signals when using CYP71B13 antibodies?

When troubleshooting weak or non-specific signals with CYP71B13 antibodies:

• Optimize antibody concentration:

  • Perform titration experiments with different antibody dilutions

  • Compare results with manufacturer's recommendations

• Modify blocking conditions:

  • Test different blocking agents (BSA, non-fat milk, commercial blockers)

  • Optimize blocking time and temperature

• Enhance signal detection:

  • Consider signal amplification methods (e.g., biotin-streptavidin systems)

  • Use more sensitive detection substrates for Western blotting

  • Increase exposure time for Western blots or imaging time for immunofluorescence

• Reduce background:

  • Increase washing stringency (more washes, longer duration, higher salt concentration)

  • Add blocking agents to antibody dilution buffer

  • Pre-adsorb antibody with plant extract from knockout/knockdown samples

• Check sample quality:

  • Ensure protein integrity by running a general protein stain

  • Verify appropriate induction conditions for CYP71B13 expression

  • Include positive controls (if available) such as recombinant CYP71B13

• Consider alternative antibody sources or lots:

  • Compare performance of antibodies from different manufacturers

  • Test different lots of the same antibody

How can CYP71B13 antibodies be used to investigate protein-protein interactions in metabolic pathways?

CYP71B13 antibodies can be powerful tools for investigating protein-protein interactions through several approaches:

• Co-immunoprecipitation (co-IP):

  • Use CYP71B13 antibodies conjugated to beads to pull down protein complexes

  • Analyze interacting partners by Western blotting with specific antibodies or by mass spectrometry

  • Include appropriate controls such as IgG or pre-immune serum

  • Consider both targeted and untargeted approaches as demonstrated in the CYP71B15 research where "untargeted co-IP approaches with the biosynthetic enzymes CYP71B15 and CYP71A13 as baits" were used to identify interacting proteins

• Proximity ligation assay (PLA):

  • Combine CYP71B13 antibody with antibodies against potential interacting partners

  • Visualize interactions in situ with subcellular resolution

• FRET-FLIM (Förster Resonance Energy Transfer-Fluorescence Lifetime Imaging Microscopy):

  • Use fluorescently labeled antibodies or fusion proteins

  • Measure energy transfer between CYP71B13 and potential interacting partners

  • This approach was successfully used to confirm interactions between CYP71A13 and other proteins where "interactions were confirmed by targeted co-IP and Förster resonance energy transfer measurements based on fluorescence lifetime microscopy (FRET-FLIM)"

• Bimolecular Fluorescence Complementation (BiFC):

  • Express CYP71B13 and potential interacting partners as fusion proteins with complementary fragments of a fluorescent protein

  • Reconstitution of fluorescence indicates protein-protein interaction
    These methods can help identify whether CYP71B13 participates in metabolons similar to those observed with other CYP71 family members in camalexin biosynthesis.

What strategies can be employed to study CYP71B13 localization and dynamics in living plant cells?

To study CYP71B13 localization and dynamics in living plant cells:

• Fluorescent protein fusion constructs:

  • Create C- or N-terminal fusions of CYP71B13 with fluorescent proteins (GFP, YFP, mCherry)

  • Express under native or inducible promoters

  • Validate functionality of fusion proteins through complementation of knockout mutants

  • Similar approaches were successful with CYP71B15 where researchers "generated a construct expressing CYP71B15 as a C-terminal GFP fusion protein under the control of its own promoter"

• Live-cell imaging techniques:

  • Confocal microscopy for high-resolution imaging

  • Spinning disc confocal for rapid imaging with reduced photobleaching

  • Super-resolution microscopy for detailed subcellular localization

• Colocalization studies:

  • Use established organelle markers (e.g., ER, Golgi, plasma membrane)

  • Quantify colocalization with appropriate statistical methods

  • CYP71B15, CYP71A12, and CYP71A13 were all found to be "localized to the ER and showed colocalization with the ER lumenal marker RFP-HDEL"

• Dynamic studies:

  • Fluorescence recovery after photobleaching (FRAP) to study protein mobility

  • Photoactivation or photoconversion to track protein movement

  • Time-lapse imaging to monitor changes in response to stimuli

• Inducible expression systems:

  • Use pathogen treatment, UV irradiation, or chemical inducers to trigger expression

  • Monitor spatiotemporal changes in localization and abundance

  • For CYP71B15, researchers observed that the protein "was only observed in cells in close proximity to successful pathogen infection"

How can researchers investigate the enzyme kinetics and substrate specificity of CYP71B13 using antibody-based approaches?

Investigating enzyme kinetics and substrate specificity of CYP71B13 using antibody-based approaches:

• Immunopurification for in vitro assays:

  • Use CYP71B13 antibodies to immunopurify the native enzyme from plant tissues

  • Conduct in vitro enzyme assays with purified enzyme and potential substrates

  • Analyze reaction products by chromatography and mass spectrometry

• Activity-based protein profiling:

  • Use activity-based probes that bind to active CYP71B13

  • Combine with CYP71B13 antibodies for specific detection and quantification

  • Compare enzyme activity under different conditions or in different genetic backgrounds

• Substrate protection assays:

  • Test if binding of potential substrates protects specific epitopes from antibody recognition

  • Use this to infer substrate binding sites and assess substrate affinity

• Reconstitution experiments:

  • Immunopurify CYP71B13 along with potential metabolon components

  • Assess how the presence of interacting partners affects substrate affinity and catalytic efficiency

  • Similar approaches revealed "increased substrate affinity of CYP79B2 in the presence of CYP71A13, indicating an allosteric interaction"

• In situ activity assays:

  • Combine immunolocalization with activity-based staining

  • Correlate enzyme localization with sites of metabolite production

How can transcriptomic data complement CYP71B13 antibody studies in understanding gene function?

Integrating transcriptomic data with CYP71B13 antibody studies:

• Correlation analysis:

  • Compare CYP71B13 transcript levels (RNA-seq, qRT-PCR) with protein levels (Western blot, ELISA)

  • Identify potential post-transcriptional regulation mechanisms

  • Similar analyses for other CYP enzymes showed that "gene expression and protein expression of CD73 were well-correlated"

• Co-expression network analysis:

  • Identify genes with expression patterns similar to CYP71B13

  • Use antibodies to validate co-expression at the protein level

  • Infer potential functional relationships or metabolic pathways

• Expression under stress conditions:

  • Monitor transcript and protein levels under various biotic and abiotic stresses

  • Identify conditions that induce CYP71B13 expression

  • Similar to observations with CYP71A12 and CYP71A13 where "pathogen infection or treatment with high dosages of UV light or heavy metals, such as silver nitrate, induce the production"

• Temporal dynamics:

  • Track the time course of gene and protein expression following induction

  • Determine if there are delays between transcriptional activation and protein accumulation

• Tissue-specific expression:

  • Compare transcriptome data from different tissues with immunolocalization studies

  • Identify tissues with high CYP71B13 expression for focused functional studies

What considerations are important when designing knockout/knockdown experiments validated with CYP71B13 antibodies?

When designing knockout/knockdown experiments that will be validated with CYP71B13 antibodies:

• Knockout strategy selection:

  • CRISPR/Cas9 for precise gene editing

  • T-DNA insertion lines if available

  • Consider potential functional redundancy with other CYP71 family members

• Knockdown approaches:

  • RNAi constructs targeting CYP71B13-specific sequences

  • Virus-induced gene silencing (VIGS) for transient knockdown

  • Inducible artificial microRNA systems for controlled reduction of expression

• Validation of genetic modifications:

  • Use CYP71B13 antibodies to confirm absence (knockout) or reduction (knockdown) of protein

  • Quantify protein levels by Western blot and compare with wild-type controls

  • Include appropriate controls such as complementation lines expressing CYP71B13 under native or constitutive promoters

• Phenotypic analysis:

  • Monitor plant development, stress responses, and metabolite profiles

  • Use CYP71B13 antibodies for immunohistochemistry to correlate phenotypes with changes in protein localization or abundance

  • Consider redundancy or compensation by related enzymes

• Experimental design considerations:

  • Include time-course studies to capture dynamic responses

  • Test multiple stress conditions to identify specific phenotypes

  • Use appropriate statistical analysis to account for biological variability

How can CYP71B13 antibodies contribute to understanding evolutionary relationships among cytochrome P450 enzymes?

CYP71B13 antibodies can provide valuable insights into evolutionary relationships among cytochrome P450 enzymes:

• Cross-reactivity studies:

  • Test CYP71B13 antibodies against homologous proteins from related plant species

  • Identify conserved epitopes that may indicate functional importance

  • Use epitope mapping to determine which protein domains are most conserved

• Comparative immunoprecipitation:

  • Use CYP71B13 antibodies to pull down homologous proteins from different species

  • Analyze by mass spectrometry to identify sequence variations and conservation

  • Compare interacting partners across species to identify conserved protein complexes

• Structural biology applications:

  • Use antibodies to stabilize CYP71B13 for crystallization attempts

  • Determine if antibody binding sites correspond to functionally important domains

  • Compare with structural data from other CYP enzymes to identify conserved features

• Functional conservation assessment:

  • Use antibodies to track expression patterns of CYP71B13 homologs in different species

  • Determine if expression is induced by similar stimuli across species

  • Correlate with metabolite profiles to assess functional conservation

What are the major challenges in developing highly specific monoclonal antibodies against CYP71B13?

Developing highly specific monoclonal antibodies against CYP71B13 faces several challenges:

• Sequence similarity with other CYP enzymes:

  • High homology between CYP71B13 and related family members can lead to cross-reactivity

  • Requires careful epitope selection focusing on unique regions

  • May need extensive validation to confirm specificity

• Protein conformation:

  • Native membrane-bound cytochrome P450 enzymes have specific conformations that may be lost during immunization with peptides or recombinant proteins

  • Conformational epitopes may be critical for specificity but difficult to preserve

  • Consider using cell-derived vesicles or exosomes containing natively folded protein as immunogens, similar to the approach where "an anti-CD73 antibody could be generated by exosome immunization"

• Post-translational modifications:

  • Plant-specific modifications may be absent in recombinant proteins produced in bacterial systems

  • Consider expression systems that maintain relevant modifications

  • Plant-based expression systems may be advantageous, as demonstrated in the production of Pembrolizumab in Nicotiana benthamiana

• Antibody validation:

  • Limited availability of verified knockout or knockdown plant materials as negative controls

  • Lack of standardized protocols for cytochrome P450 antibody validation

  • Need for multiple complementary approaches to confirm specificity

• Low natural abundance:

  • CYP71B13 may be expressed at low levels or only under specific conditions

  • May require enrichment strategies or induction before immunization or testing

How might new antibody engineering technologies improve CYP71B13 research tools?

Emerging antibody engineering technologies that could benefit CYP71B13 research:

• Single-domain antibodies (nanobodies):

  • Smaller size allows better access to cryptic epitopes

  • Potential for improved penetration in tissue samples

  • Can be expressed in plant cells as intrabodies to track or modulate CYP71B13 function in vivo

• Recombinant antibody fragments:

  • Fab, scFv, or diabody formats for improved tissue penetration

  • Can be expressed in plant systems for cost-effective production

  • May allow for site-specific labeling with fluorophores or enzymes

• Bispecific antibodies:

  • Simultaneous binding to CYP71B13 and interacting partners

  • Useful for studying protein-protein interactions in complexes or metabolons

  • Could help identify transient interactions that are difficult to capture with conventional methods

• Antibody-based biosensors:

  • Integration with FRET pairs to create conformation-sensitive reporters

  • Development of antibody-based sensors for metabolites produced by CYP71B13

  • Real-time monitoring of enzyme activity in living cells

• Plant-produced antibodies:

  • Expression of anti-CYP71B13 antibodies in plants using transient expression systems

  • Can provide cost-effective production with plant-specific glycosylation

  • Similar to the approach used for Pembrolizumab production in Nicotiana benthamiana where researchers "explored the potential of plant-based system to produce an anti-human PD-1 monoclonal antibody"

What novel insights might be gained from combining CYP71B13 antibody studies with emerging metabolomics approaches?

Integrating CYP71B13 antibody studies with metabolomics could yield several novel insights:

• Correlation between enzyme abundance and metabolite levels:

  • Quantify CYP71B13 protein using antibody-based methods (ELISA, Western blot)

  • Correlate with metabolite profiles using LC-MS/MS or other analytical techniques

  • Identify potential substrates and products based on correlative patterns

• Spatial metabolomics:

  • Combine immunohistochemistry to localize CYP71B13 with imaging mass spectrometry

  • Create spatial maps correlating enzyme localization with specific metabolites

  • Identify tissue-specific or cell-specific metabolic roles

• Immunocapture metabolomics:

  • Use CYP71B13 antibodies to isolate enzyme complexes or metabolons

  • Analyze co-purified metabolites to identify substrates, intermediates, or products

  • Detect transient or low-abundance pathway intermediates

• Stable isotope labeling:

  • Track metabolite flux in wild-type versus CYP71B13 knockout/knockdown lines

  • Use antibodies to confirm protein levels in experimental samples

  • Identify rate-limiting steps in metabolic pathways

• In situ enzyme activity visualization:

  • Develop methods to visualize enzyme activity and metabolite production simultaneously

  • Correlate with immunolocalization of CYP71B13 and potential interacting partners

  • Similar to studies of metabolon formation in camalexin biosynthesis where researchers demonstrated that the pathway is "channeled by the formation of an enzyme complex"