CYP71B37 Antibody

What is the CYP71B37 Antibody and what systems can it be used in?

CYP71B37 Antibody is a polyclonal antibody raised in rabbit against recombinant Arabidopsis thaliana CYP71B37 protein. This antibody is designed specifically for plant research applications, particularly in Arabidopsis thaliana systems. It can be used in various experimental techniques including:

• Western blotting (WB)

• Enzyme-linked immunosorbent assay (ELISA)

• Immunofluorescence/Immunocytochemistry (IF/ICC)

The antibody recognizes the native CYP71B37 protein which belongs to the cytochrome P450 superfamily, a group of enzymes involved in various metabolic processes in plants.

How should CYP71B37 Antibody be stored and handled for optimal performance?

For optimal performance and longevity of the CYP71B37 Antibody, follow these storage and handling guidelines:

When handling the antibody:

• Allow it to reach room temperature before opening the vial

• Reconstitute lyophilized antibody according to manufacturer's instructions

• Use sterile techniques when handling to prevent contamination

• Wear appropriate personal protective equipment

What are the recommended dilutions for different applications of CYP71B37 Antibody?

Based on standard protocols for similar polyclonal antibodies, the recommended working dilutions for CYP71B37 Antibody are:

These dilutions should be optimized for specific experimental conditions, including the abundance of target protein in your samples and the detection method employed .

How can I validate the specificity of CYP71B37 Antibody for my plant research?

To validate the specificity of CYP71B37 Antibody, implement a multi-step approach:

• Genetic controls: Compare wild-type plants with CYP71B37 knockout/knockdown mutants

  • If available, a CYP71B37 knockout should show significantly reduced or no signal

• Competitive inhibition assay:

  • Pre-incubate the antibody with excess purified CYP71B37 protein

  • Compare results with non-inhibited antibody

  • Specific binding should be blocked by the purified protein

• Cross-reactivity assessment:

  • Test against related CYP family members in Arabidopsis

  • Examine reactivity in other plant species with homologous proteins

• Multiple detection methods:

  • Confirm findings using alternative techniques (e.g., if using Western blot, confirm with immunoprecipitation)

  • Correlation with mRNA expression data using RT-PCR or RNA-seq

• Peptide competition assay:

  • Use the immunogenic peptide to block antibody binding

  • A significant reduction in signal indicates specificity

What are the potential experimental limitations when using CYP71B37 Antibody for studying plant stress responses?

When using CYP71B37 Antibody to study plant stress responses, researchers should be aware of several experimental limitations:

• Background and non-specific binding:

  • Plant tissues contain numerous P450 family proteins with structural similarities

  • Cross-reactivity may occur, especially in stress conditions where expression patterns change

  • Use appropriate blocking solutions and stringent washing protocols

• Stress-induced modifications:

  • Post-translational modifications during stress responses may alter epitope recognition

  • Consider complementary approaches like mass spectrometry for protein identification

• Expression level variability:

  • Stress conditions may alter expression levels unpredictably across tissues

  • Standardize sampling procedures and include multiple biological replicates

• Temporal dynamics:

  • CYP71B37 expression may fluctuate during stress response

  • Design time-course experiments to capture the full expression profile

• Technical considerations:

  • Plant tissues contain interfering compounds (phenolics, polysaccharides)

  • Optimize extraction protocols for specific plant tissues and stress conditions

  • Include appropriate extraction controls

How can I integrate CYP71B37 Antibody data with transcriptomic analyses to understand plant metabolic pathways?

To effectively integrate CYP71B37 Antibody data with transcriptomic analyses:

• Experimental design integration:

  • Design experiments to collect both protein and RNA samples from the same biological materials

  • Include multiple time points to capture dynamic regulation

  • Maintain consistent environmental conditions across all experiments

• Correlation analysis:

  • Compare protein expression (from Western blot quantification) with mRNA expression

  • Calculate Pearson or Spearman correlation coefficients

  • Investigate discrepancies that may indicate post-transcriptional regulation

• Pathway mapping:

  • Use transcriptomic data to identify co-expressed genes in the same metabolic pathway

  • Map CYP71B37 protein localization in cellular compartments using immunolocalization

  • Identify potential protein interaction partners through co-immunoprecipitation

• Data integration tools:

  • Utilize specialized software (e.g., MapMan, PathVisio) to visualize integrated data

  • Apply clustering algorithms to identify co-regulated genes and proteins

• Functional validation:

  • Design experiments to manipulate CYP71B37 expression using genetic approaches

  • Measure metabolic outputs using metabolomics

  • Connect transcriptomic changes to protein function using the antibody

What is the optimal protocol for Western blot analysis using CYP71B37 Antibody?

Detailed Western Blot Protocol for CYP71B37 Antibody:

• Sample preparation:

  • Grind plant tissue in liquid nitrogen

  • Extract proteins using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail

  • Centrifuge at 14,000 × g for 15 minutes at 4°C

  • Collect supernatant and determine protein concentration

• SDS-PAGE separation:

  • Prepare 10-12% polyacrylamide gels

  • Load 20-30 μg protein per lane

  • Run at 70V through stacking gel, then 90V through resolving gel for 2-3 hours

• Protein transfer:

  • Transfer proteins to nitrocellulose membrane at 150 mA for 60-90 minutes

  • Verify transfer using Ponceau S staining

• Blocking:

  • Block membrane with 5% non-fat milk in TBS-T for 1-1.5 hours at room temperature

• Primary antibody incubation:

  • Dilute CYP71B37 Antibody 1:500 in 5% non-fat milk/TBS-T

  • Incubate overnight at 4°C with gentle rocking

• Washing:

  • Wash membrane with TBS-T 3 times, 5 minutes each

• Secondary antibody incubation:

  • Incubate with HRP-conjugated anti-rabbit IgG (1:5000 dilution) for 1-1.5 hours at room temperature

• Detection:

  • Develop using enhanced chemiluminescence (ECL) detection

  • Expected molecular weight of CYP71B37 is approximately 58 kDa

How can I optimize immunoprecipitation protocols when using CYP71B37 Antibody?

Optimized Immunoprecipitation Protocol:

• Preparation of plant lysate:

  • Homogenize 1-2 g plant tissue in IP buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 3 mM DTT, protease inhibitor cocktail)

  • Centrifuge at 14,000 × g for 15 minutes at 4°C

  • Pre-clear lysate with 50 μl Protein A/G beads for 1 hour at 4°C

• Antibody binding:

  • Incubate 5-10 μg of CYP71B37 Antibody with 500-1000 μg of protein lysate

  • Rotate overnight at 4°C

  • For control samples, use normal rabbit IgG at the same concentration

• Immunoprecipitation:

  • Add 50 μl of pre-washed Protein A/G beads

  • Incubate for 3-4 hours at 4°C with gentle rotation

  • Collect beads by centrifugation at 3,000 × g for 1 minute

• Washing:

  • Wash beads 4-5 times with IP buffer

  • For stringent washing, include one wash with high-salt buffer (IP buffer with 300 mM NaCl)

• Elution:

  • Elute bound proteins by boiling in 50 μl of 2× SDS sample buffer for 5 minutes

  • Analyze by SDS-PAGE and Western blotting

• Optimization tips:

  • Test different antibody:protein ratios (1:50, 1:100, 1:200)

  • Compare different lysis conditions (varying detergent types and concentrations)

  • Consider crosslinking the antibody to beads to prevent antibody contamination in eluted samples

What procedures should be followed for immunohistochemistry applications with CYP71B37 Antibody?

Immunohistochemistry Protocol for Plant Tissues:

• Tissue fixation and embedding:

  • Fix plant tissues in 4% paraformaldehyde in PBS for 16-24 hours at 4°C

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

  • Embed in paraffin or optimal cutting temperature (OCT) compound

• Sectioning:

  • Cut 5-10 μm sections using a microtome

  • Mount on poly-L-lysine coated slides

  • Dry sections at 37°C overnight

• Deparaffinization and rehydration (for paraffin sections):

  • Immerse slides in xylene (2 × 10 minutes)

  • Rehydrate through decreasing ethanol series (100%, 95%, 70%, 50%, 30%)

  • Rinse in distilled water

• Antigen retrieval:

  • Heat-induced epitope retrieval: 10 mM sodium citrate buffer (pH 6.0) for 15 minutes at 95°C

  • Allow to cool to room temperature (20 minutes)

• Blocking and permeabilization:

  • Block with 5% normal goat serum, 0.3% Triton X-100 in PBS for 1 hour

• Primary antibody incubation:

  • Dilute CYP71B37 Antibody 1:50-1:200 in blocking solution

  • Incubate overnight at 4°C in a humidified chamber

• Washing:

  • Wash 3 × 5 minutes with PBS

• Secondary antibody incubation:

  • Incubate with fluorophore-conjugated anti-rabbit secondary antibody (1:200-1:500)

  • Incubate for 1-2 hours at room temperature in the dark

• Final washing and mounting:

  • Wash 3 × 5 minutes with PBS

  • Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes

  • Mount with anti-fade mounting medium

• Controls:

  • Negative control: omit primary antibody

  • Peptide competition: pre-incubate antibody with immunogenic peptide

  • Tissue specificity controls: include tissues known to express or not express CYP71B37

What are the common issues encountered with CYP71B37 Antibody in Western blotting and how can they be resolved?

When troubleshooting, systematically change one variable at a time while keeping others constant to identify the source of the problem .

How should researchers interpret contradictory results between antibody-based detection of CYP71B37 and gene expression data?

When faced with discrepancies between CYP71B37 protein detection using antibodies and corresponding gene expression data:

• Consider biological explanations:

  • Post-transcriptional regulation: miRNAs may suppress translation without affecting mRNA levels

  • Protein stability: Different half-lives of mRNA versus protein

  • Temporal dynamics: Time lag between transcription and translation

  • Subcellular localization changes: Protein may become less accessible to extraction despite high expression

• Technical considerations:

  • Antibody specificity: Verify specificity with appropriate controls

  • Sample preparation differences: Different protocols for RNA and protein extraction may affect yield

  • Detection sensitivity: qPCR may detect low-abundance transcripts not detectable at protein level

  • Normalization methods: Different normalization strategies between techniques

• Validation approaches:

  • Multiple antibodies: Test with antibodies targeting different epitopes

  • Genetic validation: Use CYP71B37 overexpression or knockout lines

  • Absolute quantification: Apply targeted proteomics (MRM-MS) for protein quantification

  • Alternative splicing analysis: Check if splice variants affect antibody recognition

• Integration strategies:

  • Correlation analysis across conditions: Plot protein vs. mRNA across multiple conditions

  • Time-course experiments: Track both parameters over time

  • Multi-omics integration: Incorporate additional layers (metabolomics, epigenetics)

What statistical approaches are most appropriate for analyzing quantitative data generated with CYP71B37 Antibody?

For robust statistical analysis of quantitative data generated with CYP71B37 Antibody:

• Experimental design considerations:

  • Minimum of 3-5 biological replicates

  • Include technical replicates for each biological sample

  • Incorporate appropriate controls (loading controls, negative controls)

  • Plan for adequate statistical power (power analysis)

• Quantification methods:

  • Densitometry analysis for Western blot bands

  • Fluorescence intensity measurement for immunofluorescence

  • Ensure linear range of detection for quantitative comparisons

• Data normalization strategies:

  • Normalize to internal controls (housekeeping proteins)

  • Consider total protein normalization (Ponceau S, SYPRO Ruby)

  • For plant studies, normalize to plant-specific reference proteins (actin, tubulin)

• Statistical tests:

  • For two-group comparisons: Student's t-test (parametric) or Mann-Whitney U test (non-parametric)

  • For multiple group comparisons: One-way ANOVA with post-hoc tests (Tukey, Bonferroni)

  • For complex designs: Two-way ANOVA, mixed-effects models

  • For correlation analysis: Pearson (linear) or Spearman (non-linear) correlation coefficients

• Advanced statistical approaches:

  • ANCOVA when controlling for covariates

  • Repeated measures ANOVA for time-course experiments

  • Multivariate analysis for complex datasets

• Reporting and visualization:

  • Include measures of dispersion (SD, SEM)

  • Report exact p-values

  • Use appropriate graphical representations (box plots for distribution, scatter plots for correlations)

  • Include confidence intervals

How can CYP71B37 Antibody be used to investigate plant secondary metabolism pathways?

CYP71B37 Antibody serves as a powerful tool for investigating plant secondary metabolism through several methodological approaches:

• Protein localization studies:

  • Determine subcellular localization of CYP71B37 using immunofluorescence microscopy

  • Map tissue-specific expression patterns using immunohistochemistry

  • This spatial information provides insights into where secondary metabolite synthesis occurs

• Protein association studies:

  • Use co-immunoprecipitation with CYP71B37 Antibody to identify protein interaction partners

  • Identify protein complexes involved in metabolic channeling of intermediates

  • These interactions may reveal novel components of metabolic pathways

• Environmental response monitoring:

  • Track changes in CYP71B37 protein levels under different environmental stressors

  • Correlate protein expression with metabolite production

  • Establish cause-effect relationships in stress-induced metabolism

• Pathway regulation studies:

  • Compare post-translational modifications under different conditions

  • Investigate how protein modification affects enzyme activity

  • Analyze protein stability and turnover rates

• Comprehensive pathway mapping:

  • Combine antibody-based protein detection with metabolomic profiling

  • Correlate enzyme abundance with specific metabolite levels

  • Establish metabolic flux models based on protein expression data

What are the emerging applications of CYP71B37 Antibody in studying plant-environment interactions?

Emerging applications of CYP71B37 Antibody in plant-environment interaction studies include:

• Climate change adaptation research:

  • Monitor CYP71B37 expression under elevated CO₂, temperature, or drought conditions

  • Correlate protein levels with adaptive metabolite production

  • Identify potential biomarkers for climate resilience

• Plant-microbe interaction studies:

  • Track CYP71B37 expression during pathogen infection or symbiotic interactions

  • Investigate protein localization at infection sites

  • Correlate with defense compound production

• Abiotic stress response mechanisms:

  • Compare CYP71B37 protein levels across different stress treatments

  • Identify stress-specific post-translational modifications

  • Develop predictive models for stress response pathways

• Nutrient response dynamics:

  • Analyze how nutrient availability affects CYP71B37 expression

  • Investigate the role in nutrient-dependent metabolite production

  • Map signaling pathways connecting nutrient sensing to metabolic responses

• Multi-stress integration:

  • Study CYP71B37 regulation under combined stresses

  • Identify regulatory hubs that integrate multiple environmental signals

  • Develop systems biology models incorporating protein-level data

How can researchers combine CYP71B37 Antibody with other molecular techniques to enhance their research outcomes?

To maximize research outcomes, researchers can integrate CYP71B37 Antibody with complementary molecular techniques:

• Integrating with genomic approaches:

  • Combine with CRISPR-Cas9 gene editing to study protein function in modified lines

  • Use chromatin immunoprecipitation sequencing (ChIP-seq) with transcription factors that regulate CYP71B37

  • Correlate genetic polymorphisms with protein expression patterns

• Proteomics integration:

  • Employ immunoprecipitation followed by mass spectrometry (IP-MS)

  • Apply proximity labeling techniques to identify nearby proteins

  • Use targeted proteomics for absolute quantification of CYP71B37 and related proteins

• Metabolomics correlation:

  • Perform parallel antibody-based protein quantification and metabolite profiling

  • Establish statistical correlations between enzyme abundance and metabolite levels

  • Develop prediction models for metabolic outputs based on protein expression

• Live-cell imaging applications:

  • Combine with fluorescent protein tagging for dynamic studies

  • Use immunofluorescence to calibrate and validate fluorescent protein fusions

  • Apply super-resolution microscopy for detailed localization studies

• Systems biology approaches:

  • Integrate antibody-derived data into computational models

  • Combine with transcriptomics, metabolomics, and phenomics data

  • Develop predictive models linking protein abundance to phenotypic outcomes

• Translational applications:

  • Apply knowledge to crop improvement strategies

  • Develop screening methods for desirable traits based on protein expression

  • Validate genetic markers through protein-level analysis