CYP71A19 Antibody

What is CYP71A19 and why is it significant in plant metabolism research?

CYP71A19 is a cytochrome P450 enzyme predominantly found in plants where it plays crucial roles in specialized metabolism pathways. As a member of the CYP71 family, this enzyme catalyzes oxidation reactions that modify chemical structures of metabolic intermediates. The significance of CYP71A19 extends to both fundamental plant biochemistry and applied agricultural research, where understanding its function can inform breeding programs for improved stress resistance or production of valuable secondary metabolites.

From a methodological perspective, antibodies against CYP71A19 enable researchers to track protein expression patterns, subcellular localization, and interaction networks. This provides critical insights into regulatory mechanisms controlling specialized metabolic pathways in different plant tissues, developmental stages, and environmental conditions.

How can I validate the specificity of a CYP71A19 antibody?

Rigorous validation is essential before conducting extensive experiments. For CYP71A19 antibodies, implement the following methodological approach:

• Western blot analysis using:

  • Recombinant CYP71A19 protein as positive control

  • Tissue lysates from plants known to express CYP71A19

  • Comparative analysis with tissues where CYP71A19 expression is absent or downregulated

  • Competition assays with the immunizing peptide

• Immunoprecipitation followed by mass spectrometry to confirm protein identity

• Immunohistochemistry with appropriate controls:

  • Pre-immune serum control

  • Secondary antibody-only control

  • Peptide competition control

Similar to validation approaches used for other CYP antibodies like CYP17A1, thorough characterization should include determination of optimal working concentrations across different applications .

What are the recommended storage conditions for maintaining CYP71A19 antibody activity?

Proper storage is critical for maintaining antibody functionality over time:

• Store concentrated antibody solutions at -20°C in small aliquots to prevent repeated freeze-thaw cycles

• Include stabilizing proteins such as BSA (0.5%) and preservatives like sodium azide (0.02%) in storage buffer

• For working solutions, maintain at 4°C for short-term use (1-2 weeks)

• Perform periodic validation tests to ensure consistent performance over time

Following storage protocols similar to those established for other CYP antibodies, proper aliquoting and storage in appropriate buffer conditions can extend shelf life to 1-2 years while maintaining reactivity and specificity .

What are the optimal conditions for using CYP71A19 antibodies in Western blotting?

Western blotting with CYP71A19 antibodies requires optimization of several parameters:

Sample Preparation:

• Use RIPA buffer supplemented with protease inhibitors for most plant tissue extractions

• For membrane-associated proteins like CYP71A19, consider specialized extraction methods to maintain protein solubility

• Heat samples at 95°C for 5 minutes in reducing sample buffer before loading

Antibody Conditions:

• Primary antibody concentration: Start with 0.1-0.3 μg/mL (similar to optimized conditions for other CYP antibodies)

• Incubation time: Overnight at 4°C often yields better results than shorter incubations

• Blocking solution: 5% non-fat dry milk in TBST is typically effective

Detection Systems:

• ECL chemiluminescence systems provide good sensitivity for most applications

• For low abundance proteins, consider enhanced chemiluminescence substrates

Expected Results:

• CYP71A19 typically appears as a band at approximately 57-58 kDa, though this may vary slightly depending on post-translational modifications (similar to other CYP proteins)

What methodologies are available for quantifying CYP71A19 protein levels in plant samples?

Several quantitative approaches can be applied to measure CYP71A19 protein levels with varying degrees of sensitivity and throughput:

Western Blot Quantification:

• Use internal loading controls (housekeeping proteins like actin or tubulin)

• Include a standard curve of recombinant CYP71A19 protein

• Apply densitometry analysis with appropriate normalization

ELISA-Based Approaches:

• Develop sandwich ELISA using CYP71A19-specific antibodies

• Indirect ELISA with proper controls and standard curves

• Competitive ELISA for higher sensitivity in complex samples

Mass Spectrometry:

• Targeted proteomics approaches using selected reaction monitoring (SRM)

• Absolute quantification using isotope-labeled peptide standards

• Relative quantification comparing specific peptide ions across samples

Each method offers distinct advantages in terms of sensitivity, specificity, and throughput, with Western blotting being most accessible for laboratories with standard equipment .

How can CYP71A19 antibodies be used effectively in immunohistochemistry of plant tissues?

Immunohistochemistry (IHC) with plant tissues presents unique challenges requiring specific methodological considerations:

Tissue Preparation:

• Fix tissues in 4% paraformaldehyde for 24 hours

• For paraffin embedding, use standard dehydration and embedding protocols

• Section thickness of 5-7 μm is typically optimal for plant tissues

Antigen Retrieval:

• Heat-induced epitope retrieval using citrate buffer (pH 6.0) is often effective

• Enzymatic retrieval may be gentler for some plant tissues sensitive to heat denaturation

Staining Protocol:

• Block endogenous peroxidases with 3% hydrogen peroxide

• Block with 2-5% normal serum from the species of the secondary antibody

• Primary antibody concentration: Start with 3-5 μg/mL (similar to protocols established for other CYP antibodies)

• Incubation time: 1-2 hours at room temperature or overnight at 4°C

• Use appropriate detection systems (ABC, HRP, or fluorescent conjugates)

For visualization, alkaline phosphatase (AP) staining systems often provide excellent signal-to-noise ratios in plant tissues, similar to those used in CYP17A1 detection protocols .

What strategies can address weak or absent signal when using CYP71A19 antibodies?

When facing detection challenges with CYP71A19 antibodies, consider these methodological solutions:

For Western Blotting:

• Increase protein loading (50-100 μg total protein may be necessary for low abundance proteins)

• Reduce stringency of washing steps (shorter washes, lower salt concentration)

• Extend primary antibody incubation time (overnight at 4°C)

• Use more sensitive detection reagents (enhanced chemiluminescence)

• Verify extraction protocol efficiency for membrane proteins like CYP71A19

For Immunohistochemistry:

• Optimize antigen retrieval methods (test both heat-mediated and enzymatic approaches)

• Increase antibody concentration incrementally (up to 10 μg/mL)

• Extend incubation time (up to 48 hours at 4°C for difficult tissues)

• Use signal amplification systems (tyramide signal amplification)

• Verify fixation has not masked the epitope

These approaches address common technical barriers in detection while maintaining experimental integrity.

How can background or non-specific binding be reduced when using CYP71A19 antibodies?

High background signals can compromise data interpretation. Address this with:

Blocking Optimization:

• Test different blocking agents (BSA, normal serum, commercial blockers)

• Increase blocking time (2-3 hours at room temperature)

• Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

Antibody Conditions:

• Dilute antibody in fresh blocking solution

• Pre-absorb antibody with tissue extracts from negative control samples

• Reduce antibody concentration incrementally

• Include 0.05-0.1% Tween-20 in antibody diluent

Washing Procedures:

• Increase number and duration of washes

• Use higher stringency wash buffers (increase salt concentration)

• Include 0.1% Tween-20 in wash buffers

These optimization strategies can significantly improve signal-to-noise ratio and experimental reliability without compromising specific detection of CYP71A19 .

How do various host species of CYP71A19 antibodies affect experimental outcomes?

The host species used for antibody production can significantly impact experimental results:

Comparative Performance:

Methodological Considerations:

• For double-labeling experiments, select antibodies from different host species

• Different host species may require different blocking agents

• Secondary antibody selection must match the host species

• Optimization parameters may differ substantially between host species

Host-specific characteristics observed with CYP17A1 polyclonal antibodies from goat hosts show lower background in some applications, which may also apply to CYP71A19 antibodies .

How can CYP71A19 antibodies be adapted for chromatin immunoprecipitation (ChIP) studies in plant systems?

While CYP71A19 is not directly a DNA-binding protein, ChIP applications may be relevant for studying transcriptional regulation:

Protocol Modifications:

• Crosslinking conditions: Standard 1% formaldehyde for 10 minutes may need optimization

• Sonication parameters: Start with 15-20 cycles (30 seconds on/off) and verify fragment size

• Antibody amount: 5-10 μg per ChIP reaction is typically required

• Protein A/G bead preparation: Pre-block with BSA and sheared salmon sperm DNA

Controls for ChIP Validation:

• Input DNA (pre-immunoprecipitation) controls

• IgG controls (non-specific immunoglobulin)

• Known negative genomic regions

• Peptide competition controls

Special Considerations for CYP Proteins:

• As membrane-associated proteins, CYP71A19 may require modified crosslinking and extraction protocols

• Consider protein-protein interaction partners that might mediate DNA associations

ChIP applications with CYP proteins require rigorous validation due to their primary function as metabolic enzymes rather than transcription factors.

What approaches can be used to study protein-protein interactions involving CYP71A19?

Understanding protein interaction networks:

Co-Immunoprecipitation (Co-IP):

• Use CYP71A19 antibodies to precipitate the protein complex

• Mild lysis conditions to preserve interactions (avoid strong detergents)

• Analysis of co-precipitated proteins by Western blot or mass spectrometry

• Reciprocal Co-IP with antibodies against suspected interaction partners

Proximity Labeling Approaches:

• BioID or TurboID fusion proteins to identify proximal proteins

• APEX2-based proximity labeling

• Analysis of biotinylated proteins by streptavidin pulldown and mass spectrometry

Fluorescence-Based Interaction Studies:

• Bimolecular Fluorescence Complementation (BiFC)

• Förster Resonance Energy Transfer (FRET)

• Fluorescence Correlation Spectroscopy (FCS)

These methods can reveal components of metabolic complexes involving CYP71A19, providing insights into its functional interactions within plant metabolism.

How can functional validation complement antibody-based studies of CYP71A19?

Antibody detection should be paired with functional studies for comprehensive characterization:

Enzyme Activity Assays:

• In vitro reconstitution with recombinant CYP71A19

• Microsomal preparations from expressing tissues

• LC-MS/MS analysis of reaction products

• Kinetic characterization with varying substrate concentrations

Genetic Approaches:

• CRISPR/Cas9 knockout or knockdown studies

• Correlation of protein levels (antibody detection) with metabolite profiles

• Phenotypic analysis of CYP71A19-modified plants

• Complementation studies to verify function

Heterologous Expression Systems:

• Yeast or bacterial expression for functional characterization

• Plant cell culture systems for native-like processing

• Correlation between expression levels (detected by antibodies) and enzyme activity

This multi-faceted approach provides stronger evidence for CYP71A19 function than antibody detection alone.

How does the performance of CYP71A19 antibodies compare with antibodies targeting related CYP proteins?

Understanding relative antibody characteristics:

Specificity Comparisons:

Performance Metrics:

• Signal-to-noise ratio comparison across different CYP antibodies

• Epitope accessibility in various applications

• Consistency across different experimental conditions

The performance characteristics of CYP71A19 antibodies generally follow patterns observed with other CYP antibodies, though epitope accessibility may differ due to protein-specific structural features .

What methodological adaptations are necessary when transitioning from other CYP antibodies to CYP71A19-specific studies?

Transitioning between research targets:

Protocol Adjustments:

• Buffer system modifications based on protein properties

• Extraction method optimization for membrane association

• Blocking agent selection based on observed background patterns

Validation Requirements:

• Parallel testing with established CYP antibodies

• Systematic optimization of key parameters

• Establishment of new baseline performance metrics

Technical Considerations:

These adaptations reflect the differences in protein properties and experimental systems between mammalian and plant CYP proteins.