CYP71A23 Antibody

What is CYP71A23 and why is it important in plant research?

CYP71A23 is a cytochrome P450 monooxygenase enzyme involved in the metabolism of various compounds in plants. Similar to other cytochrome P450 enzymes, it likely plays a role in hydroxylation reactions by inserting one oxygen atom into a substrate and reducing the second into a water molecule, with electrons provided by NADPH via cytochrome P450 reductase. Like its cytochrome P450 family members, CYP71A23 may catalyze the hydroxylation of carbon-hydrogen bonds in specific plant metabolites .

The significance of CYP71A23 lies in its potential role in specialized metabolite biosynthesis in plants, which can include defense compounds, signaling molecules, or other bioactive substances. Understanding this enzyme's function can provide insights into plant biochemical pathways, stress responses, and adaptation mechanisms. Antibodies targeting CYP71A23 serve as crucial tools for investigating its expression patterns, protein interactions, and functional roles in plant development and environmental responses.

How do I select the appropriate CYP71A23 antibody for my research?

Selecting the appropriate CYP71A23 antibody requires careful consideration of several factors:

• Specificity: Ensure the antibody specifically recognizes CYP71A23 and not other cytochrome P450 family members. Request cross-reactivity data from suppliers or test the antibody against recombinant proteins of closely related CYP enzymes.

• Host species: Consider the host species in which the antibody was raised (e.g., rabbit, mouse, goat) to avoid cross-reactivity issues in your detection system. For plant studies, rabbit polyclonal antibodies often provide good results, similar to those used for other cytochrome enzymes .

• Applications: Verify that the antibody has been validated for your specific application (Western blotting, immunohistochemistry, immunoprecipitation, etc.). For example, if you need an antibody for immunohistochemistry on plant tissues, ensure it has been tested for IHC-P applications .

• Epitope information: Review the immunogen details to understand which region of CYP71A23 the antibody targets. Antibodies raised against recombinant fragments typically covering amino acids 1-200 (N-terminal region) may provide good specificity .

• Validation data: Request validation data showing detection of both recombinant and endogenous CYP71A23 in relevant plant tissue samples.

What are the best methods for storing and handling CYP71A23 antibodies?

Optimal storage and handling of CYP71A23 antibodies are critical for maintaining their functionality:

• Storage temperature: Store antibodies at -20°C for long-term storage or at 4°C for short-term use (1-2 weeks).

• Aliquoting: Upon receipt, divide the antibody into small aliquots to minimize freeze-thaw cycles. Each freeze-thaw cycle can reduce antibody activity by approximately 10-15%.

• Buffer conditions: Maintain antibodies in appropriate buffer systems, typically containing:

  • 50-100 mM phosphate or Tris buffer (pH 7.4-8.0)

  • 150 mM NaCl

  • 0.02-0.05% sodium azide as a preservative

  • Optional: 50% glycerol for freeze protection

• Handling precautions:

  • Avoid repeated freeze-thaw cycles

  • Never vortex antibodies; mix gently by inversion or flicking

  • Keep antibodies on ice during experimental preparations

  • Use sterile technique when handling antibody solutions

  • Avoid exposure to strong light, especially for fluorophore-conjugated antibodies

• Working dilutions: Prepare working dilutions immediately before use rather than storing diluted antibodies for extended periods.

What is the optimal protocol for Western blot analysis using CYP71A23 antibody?

The following protocol has been optimized for Western blot analysis of CYP71A23 in plant tissues:

• Sample preparation:

  • Extract total protein from plant tissue using a buffer containing:

    • 50 mM Tris-HCl (pH 7.5)

    • 150 mM NaCl

    • 1% Triton X-100

    • 0.5% sodium deoxycholate

    • Protease inhibitor cocktail

  • Homogenize tissue on ice, centrifuge at 14,000 × g for 15 minutes at 4°C

  • Collect supernatant and determine protein concentration

• SDS-PAGE separation:

  • Load 20-50 μg of total protein per lane

  • Use 10-12% polyacrylamide gels for optimal resolution of CYP71A23 (predicted molecular weight ~55-60 kDa)

  • Include positive and negative controls

• Transfer conditions:

  • Transfer to PVDF membrane at 100V for 1 hour or 30V overnight at 4°C

  • Verify transfer efficiency with Ponceau S staining

• Blocking and antibody incubation:

  • Block membrane with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Incubate with primary CYP71A23 antibody at 1:1000 to 1:2000 dilution in blocking buffer overnight at 4°C

  • Wash 3 × 10 minutes with TBST

  • Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

  • Wash 3 × 10 minutes with TBST

• Detection:

  • Apply ECL substrate and develop using an imaging system

  • Exposure times typically range from 30 seconds to 5 minutes depending on expression levels

• Validation:

  • Strip and reprobe the membrane with an anti-actin or anti-GAPDH antibody as a loading control

How can I perform immunolocalization of CYP71A23 in plant tissues?

For successful immunolocalization of CYP71A23 in plant tissues, follow this optimized protocol:

• Tissue fixation and embedding:

  • Fix fresh plant tissue in 4% paraformaldehyde in PBS (pH 7.4) for 4-6 hours at 4°C

  • Wash with PBS 3 × 10 minutes

  • Dehydrate through an ethanol series (30%, 50%, 70%, 85%, 95%, 100%)

  • Clear with xylene and embed in paraffin

• Sectioning:

  • Cut 5-10 μm sections using a microtome

  • Mount sections on poly-L-lysine coated slides

  • Dry overnight at 37°C

• Antigen retrieval:

  • Deparaffinize sections in xylene and rehydrate through decreasing ethanol series

  • Perform heat-induced epitope retrieval using 10 mM sodium citrate buffer (pH 6.0) for 20 minutes

• Immunostaining:

  • Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

  • Block non-specific binding with 5% normal serum in PBS for 1 hour

  • Incubate with primary CYP71A23 antibody (1:100 to 1:500 dilution) overnight at 4°C

  • Wash 3 × 5 minutes with PBS

  • Incubate with fluorophore-conjugated secondary antibody (1:500) for 1-2 hours at room temperature

  • Wash 3 × 5 minutes with PBS

  • Counterstain nuclei with DAPI if desired

  • Mount with anti-fade mounting medium

• Imaging:

  • Capture images using confocal microscopy with appropriate excitation/emission settings

  • Include negative controls (sections incubated without primary antibody) to assess background fluorescence

• Colocalization studies:

  • For subcellular localization, consider co-staining with organelle-specific markers (e.g., ER, Golgi, or plasma membrane markers)

What controls should be included when using CYP71A23 antibody in immunoassays?

Rigorous control experiments are essential for interpreting results obtained with CYP71A23 antibody:

• Positive controls:

  • Recombinant CYP71A23 protein (if available)

  • Tissue samples known to express high levels of CYP71A23

  • Overexpression systems (e.g., plants or cell cultures transiently expressing CYP71A23)

• Negative controls:

  • Primary antibody omission (to assess secondary antibody specificity)

  • Isotype control (using non-specific IgG from same species as primary antibody)

  • Tissues from CYP71A23 knockout or knockdown plants (if available)

  • Pre-absorption control (pre-incubating antibody with excess antigen)

• Specificity controls:

  • Western blot showing single band at expected molecular weight

  • Testing cross-reactivity with recombinant proteins of closely related CYP family members

  • Testing antibody on tissues from different plant species with varying levels of sequence conservation

• Loading/processing controls:

  • Housekeeping proteins (actin, GAPDH, tubulin) for Western blots

  • General protein stains for total protein normalization

  • Constitutively expressed fluorescent proteins for microscopy samples

• Signal validation:

  • Use of multiple antibodies targeting different epitopes of CYP71A23

  • Correlation with mRNA expression data

How can I use CYP71A23 antibody for protein-protein interaction studies?

CYP71A23 antibody can be effectively utilized for investigating protein-protein interactions through several techniques:

• Co-immunoprecipitation (Co-IP):

  • Prepare plant tissue lysate in a non-denaturing buffer (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, protease inhibitors)

  • Pre-clear lysate with Protein A/G beads

  • Incubate lysate with CYP71A23 antibody (2-5 μg) overnight at 4°C

  • Add Protein A/G beads and incubate for 2-4 hours

  • Wash beads 4-5 times with buffer

  • Elute bound proteins and analyze by Western blot or mass spectrometry

  • Similar approaches have been successful for other plant proteins like VIH2

• Proximity Ligation Assay (PLA):

  • Fix and permeabilize plant tissue sections or protoplasts

  • Block non-specific binding

  • Incubate with CYP71A23 antibody and antibody against putative interacting protein

  • Apply PLA probes with oligonucleotide-conjugated secondary antibodies

  • Perform ligation and amplification according to manufacturer's protocol

  • Visualize interaction signals by fluorescence microscopy

• Bimolecular Fluorescence Complementation (BiFC):

  • This technique doesn't directly use the antibody but can validate interactions detected by antibody-based methods

  • Clone CYP71A23 and putative partners into BiFC vectors

  • Transiently express in plant cells (e.g., protoplasts or N. benthamiana leaves)

  • Analyze fluorescence reconstitution by confocal microscopy

• Pull-down validation:

  • Express recombinant tagged CYP71A23 protein

  • Perform pull-down experiments with putative interacting proteins

  • Confirm interactions by Western blot using the CYP71A23 antibody

  • This approach has been successful for confirming protein interactions in plant systems

How do I optimize CYP71A23 antibody for chromatin immunoprecipitation (ChIP) experiments?

While CYP71A23 is not expected to directly interact with chromatin as it's an enzyme rather than a transcription factor, this question might be relevant for researchers investigating potential nuclear localization or chromatin-associated roles. For ChIP optimization:

• Cross-linking optimization:

  • Test different formaldehyde concentrations (0.75-1.5%) and incubation times (5-20 minutes)

  • For plant tissues, vacuum infiltration may improve fixation efficiency

  • Quench with glycine (final concentration 125 mM)

• Chromatin preparation:

  • Isolate nuclei from plant tissues before sonication

  • Optimize sonication conditions to generate 200-500 bp DNA fragments

  • Verify fragmentation efficiency by agarose gel electrophoresis

• Antibody validation:

  • Perform preliminary Western blot on nuclear extracts to confirm nuclear presence

  • Test different antibody amounts (2-10 μg per ChIP reaction)

  • Include IgG control and input samples

• Immunoprecipitation conditions:

  • Pre-clear chromatin with Protein A/G beads

  • Incubate chromatin with antibody overnight at 4°C

  • Add beads and incubate for 2-4 hours

  • Perform stringent washes to reduce background

• Reversal of cross-links and DNA purification:

  • Incubate samples at 65°C overnight to reverse cross-links

  • Treat with RNase A and Proteinase K

  • Purify DNA using phenol-chloroform extraction or commercial kits

• Analysis methods:

  • Perform qPCR with primers targeting regions of interest

  • Consider ChIP-seq for genome-wide binding profile analysis

What approaches can I use to study post-translational modifications of CYP71A23?

Investigating post-translational modifications (PTMs) of CYP71A23 requires specialized techniques:

• Immunoprecipitation followed by mass spectrometry:

  • Immunoprecipitate CYP71A23 from plant extracts using the specific antibody

  • Separate proteins by SDS-PAGE

  • Excise the band corresponding to CYP71A23

  • Perform in-gel digestion with trypsin

  • Analyze peptides by LC-MS/MS to identify PTMs

  • Search for common modifications such as phosphorylation, glycosylation, or ubiquitination

• Phosphorylation-specific analysis:

  • Treat samples with phosphatase inhibitors during extraction

  • Use Phos-tag™ gels for enhanced separation of phosphorylated proteins

  • Perform Western blot with CYP71A23 antibody to detect mobility shifts

  • Confirm with phospho-specific antibodies if available

• Glycosylation analysis:

  • Treat protein samples with glycosidases (PNGase F, Endo H)

  • Analyze mobility shifts by Western blot

  • Use lectins to detect specific glycan structures

• Ubiquitination studies:

  • Immunoprecipitate CYP71A23 under denaturing conditions

  • Perform Western blot with anti-ubiquitin antibodies

  • Alternatively, express tagged ubiquitin in plants and perform pull-downs

• Site-directed mutagenesis validation:

  • Once PTM sites are identified, create site-directed mutants

  • Express mutants in plant systems

  • Compare activity, localization, and stability to wild-type CYP71A23

How do I address cross-reactivity issues with CYP71A23 antibody?

Cross-reactivity is a common challenge when working with antibodies targeting cytochrome P450 family members due to sequence similarities:

• Identification of cross-reactivity:

  • Test the antibody against recombinant proteins of closely related CYP family members

  • Perform Western blot on tissues from knockout/knockdown plants if available

  • Use peptide competition assays to confirm specificity

• Mitigation strategies:

  • Antibody purification: Perform affinity purification using immobilized CYP71A23-specific peptides

  • Absorption: Pre-incubate antibody with recombinant proteins of cross-reactive CYP family members

  • Dilution optimization: Test different antibody dilutions to maximize specific signal while minimizing cross-reactivity

  • Alternative antibodies: Consider using antibodies targeting different epitopes of CYP71A23

• Confirming results with complementary approaches:

  • Correlate protein detection with gene expression data (qRT-PCR, RNA-seq)

  • Use tagged recombinant versions of CYP71A23 with tag-specific antibodies

  • Employ mass spectrometry-based identification

• Data interpretation with cross-reactivity awareness:

  • Acknowledge potential cross-reactivity in publications

  • Validate key findings with multiple independent techniques

  • Consider developing more specific monoclonal antibodies for critical applications

What are the common challenges in detecting CYP71A23 in different plant tissues?

Detection of CYP71A23 across various plant tissues presents several challenges:

• Tissue-specific expression levels:

  • CYP71A23 may be expressed at varying levels in different tissues

  • Optimization of protein extraction methods for each tissue type is crucial

  • Consider using more sensitive detection methods for tissues with low expression

• Interfering compounds:

  • Plant tissues contain various compounds that can interfere with antibody binding:

    • Phenolics: Add PVPP (polyvinylpolypyrrolidone) to extraction buffers

    • Lipids: Use detergents like Triton X-100 or NP-40 in appropriate concentrations

    • Secondary metabolites: Optimize extraction buffers with additional washing steps

• Protein modifications and isoforms:

  • Post-translational modifications may mask epitopes

  • Test multiple extraction conditions (reducing vs. non-reducing)

  • Consider native vs. denaturing conditions depending on antibody characteristics

• Tissue-specific optimization table:

How can I validate the specificity of CYP71A23 antibody in my experimental system?

Thorough validation of CYP71A23 antibody specificity is essential for reliable experimental results:

• Genetic validation approaches:

  • Test antibody on tissues from CYP71A23 knockout or knockdown plants

  • Compare detection in wild-type vs. overexpression lines

  • Use CRISPR/Cas9-edited plants with epitope modifications

• Biochemical validation approaches:

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide

  • Immunoprecipitation followed by mass spectrometry identification

  • Western blot showing single band at expected molecular weight

  • Test cross-reactivity with recombinant proteins of closely related CYP enzymes

• Orthogonal validation methods:

  • Correlate protein detection with mRNA expression (qRT-PCR, RNA-seq)

  • Use multiple antibodies targeting different epitopes

  • Compare results with GFP-tagged CYP71A23 in transgenic plants

• Validation checklist:

  • Confirm band size matches predicted molecular weight

  • Verify tissue expression pattern aligns with known transcriptional data

  • Test antibody on samples prepared under multiple conditions

  • Include appropriate positive and negative controls

  • Demonstrate consistent results across multiple biological replicates

• Epitope mapping:

  • If inconsistent results are observed, consider epitope mapping to identify the exact binding region of the antibody

  • This information can help predict potential cross-reactivity with other proteins

How can CYP71A23 antibody contribute to plant metabolomics studies?

CYP71A23 antibody can significantly enhance plant metabolomics research through several approaches:

• Correlation of enzyme levels with metabolite profiles:

  • Quantify CYP71A23 protein levels across tissues or conditions using the antibody

  • Perform parallel metabolomic analyses of the same samples

  • Identify correlations between enzyme abundance and specific metabolite accumulation

  • This approach has been successfully used to study other plant enzymes and their metabolic products

• Immunoprecipitation-based enzyme activity assays:

  • Immunoprecipitate native CYP71A23 from plant extracts

  • Perform in vitro enzyme assays with potential substrates

  • Analyze reaction products by LC-MS/MS

  • Compare activity across different plant tissues or stress conditions

• Subcellular localization and metabolic compartmentalization:

  • Use the antibody for immunolocalization studies to determine subcellular localization

  • Correlate localization with distribution of relevant metabolites

  • Investigate potential co-localization with other enzymes in the same pathway

• Protein complex analysis:

  • Use antibody for co-immunoprecipitation to identify protein complexes containing CYP71A23

  • Investigate whether complex formation affects substrate specificity or catalytic efficiency

  • Correlate complex formation with metabolite profiles

• Temporal dynamics of enzyme expression and metabolism:

  • Track CYP71A23 levels during development or stress responses

  • Correlate protein abundance dynamics with changes in metabolite levels

  • Develop predictive models linking enzyme expression to metabolic outcomes

What considerations are important when developing assays to measure CYP71A23 enzyme activity?

Developing robust assays for CYP71A23 enzyme activity requires careful consideration of several factors:

• Substrate selection and preparation:

  • Identify potential physiological substrates based on metabolomics data

  • Synthesize or purify substrates to high purity (>95%)

  • Consider substrate solubility and stability in assay conditions

  • Test multiple substrate concentrations to determine kinetic parameters

• Cofactor requirements:

  • Ensure sufficient NADPH supply (cytochrome P450 enzymes typically require NADPH as electron donor)

  • Include NADPH-regenerating system (e.g., glucose-6-phosphate/glucose-6-phosphate dehydrogenase)

  • Optimize cofactor concentrations for maximum activity

• Assay buffer optimization:

  • Test different buffer systems (typically phosphate or Tris) at pH range 7.0-8.0

  • Optimize ionic strength (50-100 mM) and salt composition

  • Include appropriate divalent cations (Mg²⁺, Mn²⁺) if required

  • Add stabilizing agents (glycerol, DTT) to maintain enzyme activity

• Detection methods:

  • Direct product measurement: HPLC, LC-MS/MS for product identification and quantification

  • Coupled assays: Measure NADPH consumption spectrophotometrically (340 nm)

  • Radiometric assays: Use radiolabeled substrates for highest sensitivity

  • Fluorescence-based assays: If products exhibit fluorescence or can be derivatized

• Controls and validations:

  • Heat-inactivated enzyme controls

  • Substrate-free and enzyme-free controls

  • Inhibitor controls (general P450 inhibitors like ketoconazole)

  • Recombinant enzyme standards for activity calibration

• Data analysis considerations:

  • Determine linear range of assay

  • Calculate kinetic parameters (Km, Vmax, kcat)

  • Compare activity across different tissues or conditions

  • Normalize activity to enzyme concentration determined by quantitative Western blot

How might CYP71A23 antibody research contribute to plant biotechnology applications?

CYP71A23 antibody research has several potential applications in plant biotechnology:

• Metabolic engineering optimization:

  • Monitor CYP71A23 protein levels in engineered plants to verify expression

  • Correlate protein abundance with production of target compounds

  • Use antibody-based assays to screen transformants for optimal enzyme expression

  • This approach has been successful with other recombinant proteins expressed in plants

• Biosynthetic pathway elucidation:

  • Identify CYP71A23 involvement in specific biosynthetic pathways

  • Use immunoprecipitation to identify interacting enzymes in metabolic pathways

  • Develop complete pathway maps for specialized metabolite production

• Protein production systems:

  • Plant molecular farming has emerged as a promising platform for recombinant protein production

  • CYP71A23 antibodies can help monitor protein expression levels

  • Optimize extraction and purification processes for maximum yield

  • Typical yields of recombinant proteins from transient expression in plants range from 1-220 μg/g fresh leaf mass

• Stress response biomarkers:

  • Track CYP71A23 levels as potential biomarkers for specific stress responses

  • Develop rapid immunoassays for early detection of plant stress

  • Use in screening programs for stress-tolerant varieties

• Protein engineering and improvement:

  • Study structure-function relationships of CYP71A23

  • Design improved variants with enhanced catalytic efficiency or altered substrate specificity

  • Validate engineered proteins using antibody-based assays for expression and stability