CYP71A26 Antibody

What is CYP71A26 and what role does it play in plant systems?

CYP71A26 belongs to the cytochrome P450 monooxygenase family, which plays crucial roles in plants' metabolic processes. Similar to other cytochrome P450 enzymes, it likely uses molecular oxygen to insert one oxygen atom into a substrate while reducing the second into a water molecule, with electrons provided by NADPH via cytochrome P450 reductase . While specific CYP71A26 functions haven't been fully characterized, cytochrome P450s in the CYP71 family are frequently involved in plant specialized metabolism, including the biosynthesis of defense compounds and phytohormones. This enzyme likely catalyzes oxidation reactions in metabolic pathways that contribute to plant immunity and stress responses, similar to other characterized plant P450s.

What applications are CYP71A26 antibodies typically used for in research?

CYP71A26 antibodies can be utilized in multiple research applications, with Western blotting (WB) and immunohistochemistry (IHC) being the most common . In Western blotting, these antibodies allow researchers to detect and quantify CYP71A26 protein expression in plant tissue extracts, providing insights into regulatory mechanisms and responses to various stimuli. For immunohistochemical applications, the antibodies enable visualization of protein localization within plant tissues, helping researchers understand where the enzyme functions at cellular and subcellular levels. Additional applications may include immunoprecipitation to study protein-protein interactions and ELISA for quantitative analysis, though validation for these specific applications would be necessary.

How should I validate the specificity of a CYP71A26 antibody before use in my experiments?

Antibody validation is critical for ensuring reliable experimental results. Begin by examining the antibody's immunogen sequence to ensure it targets a unique epitope within CYP71A26 that doesn't cross-react with other plant P450 enzymes. When working with polyclonal antibodies, which recognize multiple epitopes, carefully assess potential cross-reactivity with homologous proteins . Validate specificity through Western blotting with positive controls (tissues known to express CYP71A26) and negative controls (tissues or knockout lines lacking the protein). For definitive validation, perform experiments with recombinant CYP71A26 protein as a positive control and observe the expected molecular weight band (typically 50-60 kDa for P450 enzymes). Additionally, preabsorption tests with the immunizing peptide should abolish specific antibody binding if the antibody is truly specific.

What is the recommended protocol for using CYP71A26 antibodies in Western blotting experiments?

For optimal Western blotting results with CYP71A26 antibodies, start with proper protein extraction using a buffer containing detergents suitable for membrane-associated proteins, as P450 enzymes are typically found in the endoplasmic reticulum. Use fresh tissue samples and include protease inhibitors to prevent degradation. Separate proteins on an SDS-PAGE gel (10-12% typically works well for P450s), then transfer to a PVDF or nitrocellulose membrane . Block with 5% non-fat milk or BSA in TBST for 1-2 hours at room temperature. Incubate with the primary CYP71A26 antibody at the recommended dilution (typically 1:500-1:2000) overnight at 4°C. After washing, apply an appropriate HRP-conjugated secondary antibody against the primary antibody's host species. For visualization, use enhanced chemiluminescence and expect a band at approximately 55-60 kDa. Include positive controls and molecular weight markers to confirm target identity.

How can I use CYP71A26 antibodies to study protein-protein interactions within metabolic pathways?

To study protein-protein interactions involving CYP71A26, co-immunoprecipitation (Co-IP) provides valuable insights into the enzyme's functional partners. Begin by optimizing lysis conditions that preserve natural protein interactions while effectively extracting membrane-associated proteins like CYP71A26. Crosslinking agents may help stabilize transient interactions before extraction. Perform immunoprecipitation using CYP71A26 antibodies conjugated to protein A/G beads or magnetic beads . After washing to remove non-specific interactions, analyze the precipitated protein complexes using mass spectrometry to identify interaction partners. Additionally, reciprocal Co-IPs with antibodies against suspected interaction partners can confirm relationships. For visualization of spatial interactions, proximity ligation assays (PLA) can be employed, which use oligonucleotide-linked secondary antibodies to produce fluorescent signals when target proteins are in close proximity within cells.

What approaches can I use to characterize CYP71A26 expression patterns across different plant tissues and developmental stages?

For comprehensive characterization of CYP71A26 expression patterns, employ a multi-method approach. Begin with quantitative Western blotting using CYP71A26-specific antibodies to measure protein levels across different tissues and developmental stages . Complement protein analysis with transcript profiling via RT-qPCR to determine whether expression is transcriptionally regulated. For spatial distribution within tissues, perform immunohistochemistry on fixed tissue sections using optimized antigen retrieval methods to ensure epitope accessibility. This allows visualization of cell-specific expression patterns that may reveal functional insights. For temporal analysis during development or stress responses, collect samples at defined time points and perform both Western blotting and immunohistochemistry. The combined data from protein levels, transcript abundance, and spatial localization will provide a comprehensive understanding of CYP71A26 regulation and function throughout the plant life cycle.

How can I utilize CYP71A26 antibodies to investigate post-translational modifications of the enzyme?

Investigating post-translational modifications (PTMs) of CYP71A26 requires specialized applications of antibody-based techniques. Begin with immunoprecipitation using CYP71A26-specific antibodies to isolate the protein from plant extracts . The precipitated protein can then be analyzed by mass spectrometry to identify PTMs such as phosphorylation, glycosylation, or ubiquitination. For specific PTMs, you can perform Western blotting with dual detection - first probing with the CYP71A26 antibody, then reprobing with antibodies specific to phosphorylated residues (anti-phosphoserine/threonine/tyrosine) or other modifications. Additionally, 2D gel electrophoresis followed by Western blotting can reveal charge variants resulting from PTMs. To understand the functional significance of identified modifications, compare PTM patterns under different physiological conditions or treatments. Correlation between specific modifications and enzyme activity, stability, or localization can provide insights into regulatory mechanisms controlling CYP71A26 function.

What strategies can be employed to study CYP71A26 localization at the subcellular level using antibodies?

For precise subcellular localization of CYP71A26, immunogold electron microscopy offers the highest resolution, allowing visualization of the protein in relation to specific organelles. Prepare ultrathin sections of fixed plant tissue and incubate with CYP71A26 antibodies, followed by gold-conjugated secondary antibodies . For fluorescence microscopy approaches, perform immunofluorescence on fixed and permeabilized plant cells using CYP71A26 primary antibodies and fluorophore-conjugated secondary antibodies. Co-stain with organelle-specific markers to identify precise subcellular compartments. For living cells, consider generating fluorescent protein fusions and validating their localization patterns against immunofluorescence results with native protein. Cell fractionation followed by Western blotting with CYP71A26 antibodies provides biochemical confirmation of localization, complementing microscopy data. Since P450 enzymes typically localize to the endoplasmic reticulum, pay particular attention to distinguishing between different ER domains and potential dynamic relocalization under different conditions.

How should I interpret Western blot data when unexpected band patterns appear with CYP71A26 antibodies?

When encountering unexpected band patterns in Western blots with CYP71A26 antibodies, systematic analysis is essential. First, verify that positive controls show the expected band at approximately 55-60 kDa . Multiple bands may result from several phenomena: proteolytic degradation (remedy by using fresh samples with additional protease inhibitors), splice variants (verify through RT-PCR), post-translational modifications (confirm using deglycosylation enzymes or phosphatase treatments), or cross-reactivity with related P450 enzymes (address by increasing washing stringency or using a more specific antibody). Bands significantly larger than expected might indicate protein aggregation (add reducing agents) or homo/heterodimer formation (use non-reducing conditions to preserve). Optimizing blocking conditions and antibody dilutions can also improve specificity. If persistent non-specific bands occur, preabsorb the antibody with the immunizing peptide to identify which bands represent specific binding. Document all optimization steps and consistently apply successful protocols across experiments to ensure reproducibility.

What statistical approaches are most appropriate for quantifying CYP71A26 expression across experimental conditions?

For robust quantification of CYP71A26 expression, employ appropriate statistical methods tailored to your experimental design. Begin by ensuring proper experimental setup with sufficient biological replicates (minimum n=3, preferably n≥5) and technical replicates of Western blots or immunohistochemistry analyses . For Western blot quantification, normalize CYP71A26 band intensities to appropriate loading controls (GAPDH, actin, or total protein staining), and analyze using densitometry software. Apply log transformation if data shows heteroscedasticity. For comparing expression across multiple conditions, use ANOVA followed by appropriate post-hoc tests (Tukey's HSD for all pairwise comparisons or Dunnett's test when comparing against a control group). For immunohistochemistry quantification, analyze staining intensity across defined tissue regions using appropriate imaging software, and apply similar statistical approaches. When analyzing correlations between CYP71A26 expression and physiological parameters, use regression analysis or Pearson/Spearman correlation coefficients depending on data distribution. Report effect sizes alongside p-values to indicate biological significance beyond statistical significance.

How can I distinguish between specific and non-specific binding in immunohistochemical staining with CYP71A26 antibodies?

Distinguishing specific from non-specific binding in immunohistochemistry requires rigorous controls and optimization. Always include a negative control by omitting the primary antibody while maintaining identical secondary antibody and detection conditions, which reveals background staining . Include biological negative controls using tissues known not to express CYP71A26 or knockout/knockdown lines when available. Perform peptide competition assays by pre-incubating the antibody with excess immunizing peptide, which should eliminate specific staining while non-specific binding remains. Optimize fixation protocols to preserve antigen integrity while maintaining tissue morphology - overfixation may mask epitopes, while underfixation compromises structural integrity. Test multiple antibody dilutions to determine the optimal concentration that maximizes specific signal while minimizing background. When possible, correlate immunohistochemistry results with gene expression data to confirm biological plausibility of observed staining patterns. If persistent background occurs, modify blocking solutions (try different proteins or concentrations) and increase washing stringency (longer washes, higher salt concentrations, or addition of detergents like Tween-20).

How can CYP71A26 antibodies be used in combination with mass spectrometry for comprehensive protein analysis?

Integrating CYP71A26 antibodies with mass spectrometry creates powerful approaches for detailed protein characterization. Immunoprecipitation with CYP71A26 antibodies followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) allows identification of protein interaction partners within metabolic complexes . For analyzing post-translational modifications, enrich CYP71A26 protein through immunoprecipitation, digest with proteases, and analyze resulting peptides by LC-MS/MS to map modification sites with residue-level precision. Multiple reaction monitoring (MRM) mass spectrometry combined with isotope-labeled peptide standards enables absolute quantification of CYP71A26 protein levels across samples. For studying protein dynamics, combine pulse-chase experiments with immunoprecipitation and MS analysis to determine protein turnover rates. When analyzing complex samples, use CYP71A26 antibodies for Western blotting validation of mass spectrometry findings, creating complementary lines of evidence. This integrated approach enables comprehensive characterization of CYP71A26 abundance, interactions, modifications, and dynamics within plant cellular contexts.

What approaches can be used to study the relationship between CYP71A26 genetic variants and protein expression or function?

To investigate relationships between CYP71A26 genetic variants and protein characteristics, employ a multi-faceted approach combining genetic and protein analysis. First, identify natural variants or create engineered mutations in the CYP71A26 coding sequence, focusing on conserved domains or predicted functional regions . Express these variants in heterologous systems and quantify protein levels using Western blotting with CYP71A26 antibodies to assess effects on protein stability. Perform immunolocalization to determine if variants affect subcellular targeting. For functional analysis, conduct enzyme activity assays with purified variant proteins and compare kinetic parameters. Use structural modeling informed by antibody epitope mapping to predict how variants might affect protein folding or substrate binding. In plant systems, complement these approaches with phenotypic analysis of variant lines, correlating CYP71A26 protein levels (measured by quantitative Western blotting) with metabolite profiles and physiological responses. This integrated approach connects genetic variation to protein expression, localization, and function, providing mechanistic insights into CYP71A26's role in plant metabolism.

How can I develop a high-throughput immunoassay for quantifying CYP71A26 across large sample sets?

Developing a high-throughput immunoassay for CYP71A26 quantification requires careful optimization for specificity, sensitivity, and reproducibility. Begin by selecting a format - sandwich ELISA offers superior specificity but requires two antibodies recognizing different epitopes, while indirect ELISA is simpler but may have lower specificity . For sandwich ELISA, use a capture antibody against CYP71A26, followed by sample addition and detection with a different CYP71A26 antibody conjugated to an enzyme or fluorophore. Optimize protein extraction protocols to efficiently solubilize the membrane-associated CYP71A26 while minimizing interfering compounds. Develop a standard curve using recombinant CYP71A26 protein at known concentrations. For high-throughput applications, adapt the optimized protocol to 384-well plate format and integrate with automated liquid handling systems. Implement rigorous quality control measures, including technical replicates, inter-plate calibration standards, and regular verification with Western blotting. For specialized needs, consider developing a multiplex assay that simultaneously quantifies CYP71A26 alongside other proteins of interest using differentially labeled antibodies or bead-based platforms, enabling comprehensive protein profiling across large experimental designs.

What strategies can be employed to use CYP71A26 antibodies for investigating protein dynamics during stress responses?

To investigate CYP71A26 dynamics during stress responses, develop time-course experiments capturing both rapid and long-term changes. Begin with a carefully designed sampling strategy capturing relevant timepoints based on known stress response phases . Use quantitative Western blotting with CYP71A26 antibodies to track protein abundance changes, ensuring consistent extraction efficiency by normalizing to appropriate controls. Complement protein quantification with transcript analysis to determine whether changes occur at transcriptional or post-transcriptional levels. For spatial dynamics, perform immunohistochemistry at key timepoints to visualize potential redistribution of expression across tissues or cell types. To capture rapid protein modification events, employ phospho-specific antibodies or total CYP71A26 immunoprecipitation followed by PTM analysis. For protein stability assessment, perform cycloheximide chase experiments combined with Western blotting to determine if stress alters protein turnover rates. Integrate these approaches with metabolomic analysis to correlate CYP71A26 dynamics with changes in substrate or product metabolites. This comprehensive approach reveals mechanisms regulating CYP71A26 during stress adaptation, potentially identifying control points for enhancing plant resilience.

How might single-cell analysis techniques be combined with CYP71A26 antibodies to reveal cell-type specific expression patterns?

Single-cell analysis combined with CYP71A26 antibodies offers unprecedented resolution of expression heterogeneity within plant tissues. For tissue-based approaches, employ multiplexed immunofluorescence with CYP71A26 antibodies alongside cell-type markers, analyzing through high-resolution confocal microscopy and quantitative image analysis . Flow cytometry or FACS with CYP71A26 antibodies allows quantitative analysis of protein levels in protoplasts derived from different tissues, though careful optimization is needed for membrane protein detection. For highest resolution, consider mass cytometry (CyTOF) using metal-conjugated CYP71A26 antibodies, enabling simultaneous detection of multiple proteins in single cells without spectral overlap concerns. Alternatively, integrate spatial transcriptomics with immunohistochemistry on sequential sections to correlate CYP71A26 protein localization with comprehensive transcriptional profiles. For living tissue imaging, develop a strategy using cell-permeable CYP71A26 antibody fragments combined with proximity ligation assays. These approaches reveal whether specific cell types or developmental states exhibit distinct CYP71A26 expression patterns, potentially identifying specialized metabolic capacities within complex tissues.