CYP71B29 Antibody

What is CYP71B29 and why is it important in plant research?

CYP71B29 (UniProt AC: Q9SAE4) is a cytochrome P450 family protein found in Arabidopsis thaliana, also known as the mouse-ear cress . It is encoded by the gene At1g13100 and belongs to the broader cytochrome P450 71B subfamily. This protein is significant in plant research because cytochrome P450 enzymes play critical roles in plant metabolism, particularly in the biosynthesis of secondary metabolites, hormone metabolism, and detoxification processes. Understanding CYP71B29's function contributes to our knowledge of plant biochemical pathways and stress responses. While CYP71B29 is distinct from the related CYP71 protein, research on the CYP71 family provides valuable context for understanding its potential functions .

How do researchers validate the specificity of CYP71B29 antibodies?

Researchers typically validate CYP71B29 antibody specificity through multiple complementary approaches:

• Western blot analysis against both recombinant CYP71B29 protein and plant tissue extracts, comparing wild-type and knockout/knockdown lines

• Immunoprecipitation followed by mass spectrometry to confirm pulldown of the target protein

• Cross-reactivity testing against related cytochrome P450 family members

• Peptide competition assays where pre-incubation with the immunizing peptide should abolish signal

For optimal validation, researchers should perform apparent affinity measurements in cell-based formats, as these can reveal important avidity effects not detected in protein-based assays . The apparent Kᴅ values should be determined through flow cytometry using an eleven-point dilution series of the antibody. Discrepancies between protein-based and cell-based affinity measurements are common due to these avidity effects in the cellular context .

What expression patterns have been documented for CYP71B29 in different plant tissues?

CYP71B29 expression varies across different tissues and developmental stages in Arabidopsis thaliana. Current research has documented expression patterns through both transcriptomic and proteomic approaches. The protein's presence can be detected through immunohistochemistry using validated CYP71B29-specific antibodies. While detailed expression data specific to CYP71B29 is still emerging, members of the cytochrome P450 71B subfamily typically show tissue-specific expression patterns and are often induced under various stress conditions. Research using immunolabeling techniques with anti-CYP71B29 antibodies has been instrumental in characterizing its spatial and temporal distribution patterns in plant tissues, providing insights into its potential physiological functions.

What are the recommended approaches for generating CYP71B29-specific antibodies?

For generating highly specific CYP71B29 antibodies, researchers should consider these methodological approaches:

• Antigen selection: Target unique epitopes in non-conserved regions of CYP71B29 to minimize cross-reactivity with other P450 family members. Perform sequence alignment analysis to identify CYP71B29-specific regions.

• Production strategies:

  • Peptide antibodies: Synthesize unique peptide sequences (15-20 amino acids) from CYP71B29

  • Recombinant protein: Express partial domains that lack conserved P450 motifs

  • Genetic immunization: Use DNA constructs encoding unique CYP71B29 regions

• Host selection: Consider rabbits for polyclonal production or mice/rats for monoclonal antibody development depending on research needs.

• Post-production purification: Implement affinity purification against the immunizing antigen to enhance specificity and reduce background.

When evaluating antibody quality, researchers should determine both protein-based affinity (recombinant protein binding) and cell-based apparent affinity measurements, recognizing that discrepancies between these values (as observed with other antibodies with Kᴅ values of 0.6-3.7 nM in cell-based assays) are expected due to avidity effects in cellular contexts .

How can researchers determine if their CYP71B29 antibody maintains functionality after fluorophore conjugation?

To ensure that CYP71B29 antibodies maintain functionality after fluorophore conjugation:

• Pre- and post-conjugation affinity comparison: Measure binding affinities before and after dye conjugation using surface plasmon resonance (SPR) or bio-layer interferometry (BLI). Similar to documented cases with other antibodies, expect minimal affinity disruption if the conjugation is properly executed .

• Binding validation protocol:

  • Perform binding assays with recombinant CYP71B29 protein

  • Compare cell-based apparent affinity measurements between unconjugated and conjugated antibodies

  • Expect some variation between protein-based (Kᴅ) and cell-based apparent affinity measurements due to avidity effects

• Dye-to-protein ratio (DPR) optimization:

  DPR Range	Expected Outcome	Recommended Applications
  2-4	Minimal impact on binding	Most applications
  5-7	Moderate impact	Flow cytometry, high-signal applications
  >8	Significant impact possible	Not recommended

• Functional validation: Perform immunoprecipitation experiments with the conjugated antibody followed by mass spectrometry to confirm that target recognition remains specific and efficient.

The degree of fluorophore labeling should be determined through SDS-PAGE analysis of the dye-conjugated antibody with visualization on appropriate imaging systems (e.g., Typhoon scanner with appropriate laser/filter combinations for the specific fluorophore) .

What quality control parameters are essential before using CYP71B29 antibodies in critical experiments?

Before employing CYP71B29 antibodies in crucial experiments, researchers should verify these essential quality control parameters:

• Specificity validation:

  • Western blot analysis showing a single band at the expected molecular weight (~55 kDa for CYP71B29)

  • Absence of signal in CYP71B29 knockout/knockdown lines

  • Immunoprecipitation followed by mass spectrometry confirmation

• Sensitivity assessment:

  • Limit of detection determination using purified recombinant protein

  • Signal-to-noise ratio evaluation in relevant biological samples

• Reproducibility testing:

  • Lot-to-lot consistency verification through standardized assays

  • Stability assessment under experimental storage conditions

• Cross-reactivity profiling:

  • Testing against closely related cytochrome P450 family members

  • Evaluation in tissue panels from species where cross-reactivity might occur

• Application-specific validation:

  Application	Specific Validation Methods
  Western blot	Concentration optimization, blocking condition assessment
  Immunoprecipitation	Pull-down efficiency quantification
  Immunohistochemistry	Fixation protocol optimization, background minimization
  Flow cytometry	Titration curves, compensation controls

Researchers should maintain detailed records of these validation experiments to ensure experimental reproducibility and reliable interpretation of results with CYP71B29 antibodies.

How should researchers optimize immunoprecipitation protocols for CYP71B29?

Optimizing immunoprecipitation (IP) protocols for CYP71B29 requires systematic methodology refinement:

• Lysis buffer optimization:

  • Start with plant-specific extraction buffers containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100

  • Add protease inhibitors, 1 mM DTT, and 1 mM EDTA to preserve protein integrity

  • For membrane-associated fractions, consider including 0.1% SDS or 0.5% sodium deoxycholate

• Antibody binding conditions:

  • Test both direct antibody coupling to beads and indirect capture using Protein A/G

  • Optimize antibody:lysate ratio (typically starting at 2-5 μg antibody per 500 μg total protein)

  • Determine optimal incubation time and temperature (4°C overnight versus shorter room temperature incubations)

• Washing stringency balance:

  Wash Buffer Composition	Application	Consideration
  Low stringency: 150 mM NaCl, 0.1% detergent	Preserving weak interactions	Higher background
  Medium stringency: 300 mM NaCl, 0.1-0.5% detergent	Standard protocols	Balanced approach
  High stringency: 500 mM NaCl, 0.5-1% detergent	Removing nonspecific binding	May disrupt legitimate interactions

• Elution strategy selection:

  • Peptide competition (gentle, preserves activity)

  • Low pH glycine buffer (efficient, may affect protein structure)

  • SDS sample buffer (complete elution for downstream analysis)

• Validation approaches:

  • Western blot confirmation of target protein

  • Mass spectrometry analysis to confirm identity and identify interaction partners

  • Reverse IP with identified partners to confirm interactions

When developing co-IP protocols to study protein-protein interactions, researchers should consider that CYP71B29 may interact with proteins involved in phase separation mechanisms similar to those observed with the related CYP71 in miRNA processing pathways .

What are the best practices for using CYP71B29 antibodies in immunohistochemistry/immunofluorescence?

For optimal results with CYP71B29 antibodies in plant tissue immunohistochemistry/immunofluorescence:

• Fixation protocol optimization:

  • Compare different fixatives: 4% paraformaldehyde (standard), Carnoy's solution (better for nuclear proteins), or glutaraldehyde (enhanced membrane preservation)

  • Determine optimal fixation time (typically 2-24 hours) based on tissue type and thickness

  • Consider epitope accessibility: some fixatives may mask the CYP71B29 epitope

• Antigen retrieval methods:

  • Heat-induced epitope retrieval: citrate buffer (pH 6.0) at 95°C for 10-20 minutes

  • Enzymatic retrieval: proteinase K (1-5 μg/ml) for membrane-associated proteins

  • For recalcitrant plant tissues, combinatorial approaches may be necessary

• Antibody dilution and incubation conditions:

  • Perform titration experiments (typical starting range: 1:100-1:500)

  • Test both room temperature (2-4 hours) and 4°C (overnight) incubations

  • Include appropriate blocking solutions with plant-specific considerations (e.g., 5% BSA, 5% normal serum, 0.3% Triton X-100)

• Signal amplification considerations:

  • Direct detection with fluorophore-conjugated primary antibodies

  • Indirect detection with labeled secondary antibodies (higher sensitivity)

  • Tyramide signal amplification for low-abundance proteins

• Controls and validation:

  • Negative controls: secondary antibody alone, pre-immune serum, antibody pre-absorbed with immunizing peptide

  • Positive controls: tissues known to express CYP71B29

  • Genetic controls: comparison with knockout/knockdown lines

For multi-label experiments with CYP71B29 and other markers, researchers can employ dual-label internalization assays with simultaneous exposure of cells to two different antibodies conjugated with distinct fluorophores, similar to the approach validated with other antibody systems .

How can researchers measure CYP71B29 protein-protein interactions using antibody-based methods?

To investigate CYP71B29 protein-protein interactions using antibody-based approaches:

• Co-immunoprecipitation (Co-IP) optimization:

  • Use mild detergents (0.1-0.5% NP-40 or Triton X-100) to preserve protein complexes

  • Consider chemical crosslinking (0.5-2% formaldehyde for 10-15 minutes) to stabilize transient interactions

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

• Proximity ligation assay (PLA) implementation:

  • Combine CYP71B29 antibody with antibodies against suspected interaction partners

  • Optimize antibody dilutions and PLA probe concentrations

  • Include appropriate controls: omitting primary antibodies, using unrelated antibody pairs

• Förster resonance energy transfer (FRET) applications:

  • Conjugate CYP71B29 antibody with donor fluorophore (e.g., Alexa Fluor 488)

  • Conjugate partner protein antibody with acceptor fluorophore (e.g., Alexa Fluor 594)

  • Measure energy transfer through acceptor photobleaching or spectral imaging

• Bimolecular fluorescence complementation (BiFC) adaptation:

  • Design split fluorescent protein constructs fused to CYP71B29 and potential partners

  • Express in plant protoplasts or through transient transformation

  • Use antibodies to confirm expression levels of fusion proteins

• Phase separation analysis:
  Based on findings with related proteins like CYP71, which promotes phase separation of interaction partners , researchers should consider:

  Method	Application	Data Output
  Differential centrifugation	Physical separation of phase-separated complexes	Western blot quantification
  Fluorescence recovery after photobleaching (FRAP)	Dynamics of CYP71B29 in potential biomolecular condensates	Recovery curves and diffusion coefficients
  Immunofluorescence microscopy	Visualization of co-localization in potential condensates	Co-localization coefficients

When investigating potential CYP71B29 interactions with proteins involved in RNA processing pathways, researchers should consider whether CYP71B29 exhibits peptidyl-prolyl isomerase activity similar to CYP71, which could influence partner protein conformation and complex formation .

What are common challenges in CYP71B29 antibody applications and how can they be overcome?

Researchers frequently encounter these challenges when working with CYP71B29 antibodies:

• Cross-reactivity with related P450 family members:

  • Solution: Pre-absorb antibody with recombinant related P450 proteins

  • Alternative: Design peptide antibodies against unique regions identified through comprehensive sequence alignment

  • Validation: Test antibody against tissues from knockout plants for multiple P450 family members

• Low signal intensity in plant tissues:

  • Solution: Optimize extraction buffers with plant-specific considerations (higher detergent concentrations, plant protease inhibitor cocktails)

  • Alternative: Implement signal amplification systems (tyramide signal amplification, polymer-based detection)

  • Validation: Compare detection sensitivity across different tissue preparation methods

• Inconsistent immunoprecipitation results:

  • Solution: Test different antibody immobilization strategies (direct coupling vs. Protein A/G capture)

  • Alternative: Employ sandwich immunoprecipitation with two different antibodies recognizing distinct epitopes

  • Validation: Confirm pull-down efficiency through parallel Western blot analysis

• Background in immunohistochemistry:

  • Solution: Increase blocking stringency (longer blocking times, alternative blocking agents like plant-derived proteins)

  • Alternative: Implement alternative fixation protocols that better preserve epitope accessibility

  • Validation: Include absorption controls with immunizing peptide

• Antibody performance variation between applications:

  Application	Common Issue	Specialized Solution
  Western blot	Weak signal	Membrane activation with methanol, longer transfer times
  Immunofluorescence	High autofluorescence	Implement spectral unmixing, use far-red fluorophores
  Flow cytometry	Poor discrimination	Optimize cell preparation, increase antibody concentration
  ChIP applications	Low enrichment	Increase crosslinking time, optimize sonication conditions

For specialized applications like studying phase separation behavior similar to CYP71's role with SERRATE , researchers should consider developing antibodies that specifically recognize distinct conformational states of CYP71B29.

How can researchers determine if CYP71B29 undergoes post-translational modifications using antibody-based approaches?

To investigate post-translational modifications (PTMs) of CYP71B29 using antibody-based methods:

• Phosphorylation analysis:

  • Immunoprecipitate CYP71B29 using validated antibodies

  • Probe with pan-phospho antibodies (anti-pSer, anti-pThr, anti-pTyr)

  • Perform phosphatase treatment controls to confirm specificity

  • Implement Phos-tag gel electrophoresis for mobility shift detection

• Ubiquitination detection:

  • Combined immunoprecipitation approach: pull down with CYP71B29 antibody, probe with anti-ubiquitin

  • Alternative: Tandem Ubiquitin Binding Entities (TUBEs) pull-down followed by CYP71B29 detection

  • Control: Include proteasome inhibitors (MG132) to enhance detection of unstable ubiquitinated forms

• Glycosylation assessment:

  • Lectin blotting after CYP71B29 immunoprecipitation

  • Enzymatic deglycosylation (PNGase F, Endo H) followed by Western blot to detect mobility shifts

  • Control: Compare with chemically synthesized glycosylated and non-glycosylated peptide standards

• Other PTM investigations:

  • SUMOylation: Co-IP with SUMO-specific antibodies

  • Acetylation: IP-Western with anti-acetyl lysine antibodies

  • Methylation: IP-Western with methyl-arginine or methyl-lysine antibodies

• Mass spectrometry validation:

  Sample Preparation	Mass Spec Technique	PTM Information
  In-gel digestion after IP	LC-MS/MS	PTM identification and localization
  Immobilized antibody enrichment	Targeted MS	Quantitative PTM changes
  Chemical labeling (TMT, iTRAQ)	Quantitative proteomics	PTM stoichiometry across conditions

Similar to studies of cyclophilin proteins like CYP71, which exhibit peptidyl-prolyl isomerase (PPIase) activity , researchers should investigate whether CYP71B29 undergoes proline isomerization or catalyzes this modification in other proteins, potentially affecting phase separation behavior or protein complex formation.

How does CYP71B29 antibody binding kinetics affect experimental outcomes?

Understanding CYP71B29 antibody binding kinetics is crucial for experimental design and interpretation:

How can CYP71B29 antibodies be used to investigate protein phase separation similar to CYP71?

Based on findings showing CYP71's role in promoting phase separation of the SERRATE protein in plant miRNA processing , researchers can adapt similar approaches to investigate potential phase separation behavior of CYP71B29:

• Immunofluorescence-based condensate detection:

  • Use CYP71B29 antibodies to visualize potential biomolecular condensates in fixed cells

  • Implement co-staining with known phase separation markers

  • Apply quantitative image analysis to measure condensate size, number, and intensity

• Functional impact assessment:

  • Combine immunofluorescence with RNA FISH to correlate CYP71B29 condensates with RNA processing

  • Employ proximity ligation assays to detect protein-protein interactions within condensates

  • Use genetic knockdown/knockout approaches to assess CYP71B29's contribution to condensate formation

• Biochemical separation and analysis:

  • Develop differential centrifugation protocols to isolate potential CYP71B29-containing condensates

  • Use antibodies for immunoprecipitation of condensate components

  • Perform proteomic analysis to identify co-segregating factors

• Phase separation modulators:

  Modulator	Application Method	Expected Outcome if Phase Separation Occurs
  1,6-hexanediol	5-10% treatment of live cells	Disruption of condensates
  Temperature shifts	Controlled temperature changes	Reversible dissolution/formation
  Salt concentration	Buffer exchange in biochemical assays	Ionic strength-dependent behavior

• PPIase activity connection:
  Similar to CYP71's peptidyl-prolyl isomerase activity affecting SERRATE phase separation , investigate whether:

  • CYP71B29 exhibits similar enzymatic activity

  • This activity influences phase separation of interaction partners

  • PPIase inhibitors alter CYP71B29-associated condensate properties

Researchers should compare findings with CYP71B29 to documented behaviors of CYP71 to determine whether these related proteins share functional mechanisms in distinct biological pathways.

What approaches can researchers use to study CYP71B29 antibody internalization and trafficking?

For investigating CYP71B29 antibody internalization and intracellular trafficking:

• Quantitative internalization assays:

  • Adapt established flow cytometry-based internalization assays using fluorophore-conjugated CYP71B29 antibodies

  • Implement anti-Alexa Fluor antibodies to quench surface signals, allowing specific measurement of internalized antibody, similar to validated protocols for other systems

  • Use dual-label approaches with two different antibodies conjugated to distinct fluorophores for comparative studies

• Live-cell imaging protocols:

  • Employ pH-sensitive fluorophores (e.g., pHrodo) that increase fluorescence in acidic compartments

  • Implement photoactivatable or photoconvertible tags for pulse-chase visualization

  • Combine with lysosomal or endosomal markers (anti-LAMP1) to track intracellular fate

• Fixed-cell co-localization studies:

  • Perform time-course experiments with fixation at different intervals after antibody addition

  • Use markers for early endosomes (EEA1), late endosomes/lysosomes (LAMP1), and recycling endosomes (Rab11)

  • Calculate quantitative co-localization metrics (Pearson's, Manders' coefficients)

• Biochemical trafficking assessment:

  Method	Protocol Adaptation	Data Output
  Subcellular fractionation	Density gradient separation after antibody treatment	Western blot quantification across fractions
  Biotinylation pulse-chase	Surface biotinylation followed by internalization	Internalization rate measurement
  Recycling assays	Acid wash to remove surface antibody, measure reappearance	Recycling rate determination

• Mechanistic investigations:

  • Use endocytosis inhibitors (dynasore, chlorpromazine) to determine entry pathways

  • Employ lysosomal inhibitors (bafilomycin A1, chloroquine) to assess degradation fate

  • Test temperature blocks (4°C) to distinguish active transport from passive diffusion

When applying these methods, researchers should calculate internalization rates using quantitative metrics similar to those established for other antibody systems, including median fluorescence intensity measurements and determination of apparent Kd values through multi-point dilution series .

How can researchers integrate CYP71B29 antibody applications with cutting-edge plant miRNA processing research?

Building on discoveries about CYP71's role in miRNA processing through its interaction with SERRATE and promotion of phase separation , researchers can develop integrated approaches using CYP71B29 antibodies:

• Comparative functional analysis:

  • Use co-immunoprecipitation with CYP71B29 antibodies to identify potential RNA processing partners

  • Perform parallel studies with CYP71 to determine functional overlap or divergence

  • Implement genetic complementation experiments across cyp71 and cyp71b29 mutant backgrounds

• D-body localization and dynamics:

  • Employ CYP71B29 antibodies in co-localization studies with known D-body components

  • Investigate whether CYP71B29, like CYP71, influences phase separation behavior of miRNA processing factors

  • Combine with RNA visualization techniques to correlate with miRNA precursor localization

• PPIase activity assessment:

  • Develop in vitro PPIase activity assays for immunopurified CYP71B29

  • Compare catalytic parameters with those of CYP71

  • Identify potential substrate proteins containing proline residues at critical positions

• Integrated multi-omics approaches:

  Technique	Application	Integration with Antibody Methods
  RNA-seq	Transcriptome-wide effects	IP-RNA-seq with CYP71B29 antibodies
  Small RNA-seq	miRNA profile changes	Correlation with CYP71B29 expression/localization
  Proteomics	Interaction partner identification	IP-mass spectrometry with CYP71B29 antibodies
  CLIP-seq	RNA binding profiles	CYP71B29 antibody-based CLIP

• Structure-function relationships:

  • Use conformational-specific antibodies to capture distinct states of CYP71B29

  • Investigate whether PPIase activity (if present) influences protein conformation

  • Compare structural properties with CYP71 to identify conserved functional mechanisms

Researchers should place particular emphasis on determining whether CYP71B29's potential role in phase separation is dependent on PPIase activity, as has been demonstrated for CYP71 in promoting SERRATE phase separation and D-body assembly . This mechanistic insight could establish a broader paradigm for how peptidyl-prolyl isomerase activity regulates biomolecular condensate formation in plant RNA processing pathways.

What are the current limitations in CYP71B29 antibody research and future directions?

Current research on CYP71B29 antibodies faces several limitations that define future research opportunities:

• Technical limitations:

  • Limited commercial availability of validated CYP71B29-specific antibodies

  • Challenges in distinguishing between closely related cytochrome P450 family members

  • Difficulties in preserving native protein conformation during sample preparation

• Knowledge gaps:

  • Incomplete understanding of CYP71B29's physiological roles and regulation

  • Limited characterization of its interaction network and potential role in phase separation

  • Unclear relationship between CYP71B29 and the functionally characterized CYP71

• Methodological challenges:

  • Need for improved protocols for membrane protein extraction from plant tissues

  • Difficulties in achieving consistent results across different plant developmental stages

  • Limitations in detecting low-abundance protein-protein interactions

• Future research directions:

  Research Area	Specific Direction	Antibody Application
  Functional genomics	CYP71B29 conditional knockouts	Validation and phenotypic analysis
  Protein engineering	Structure-guided antibody development	Conformational-specific detection
  Systems biology	Integration with other -omics data	Multi-modal data correlation
  Evolutionary biology	Cross-species comparisons	Conserved epitope recognition

• Emerging technologies:

  • Development of nanobodies or aptamers as alternatives to conventional antibodies

  • Implementation of proximity-dependent labeling techniques (BioID, APEX)

  • Application of super-resolution microscopy for detailed localization studies