CYP735A2 Antibody

What is CYP735A2 and why is it significant in plant biology research?

CYP735A2 is a gene encoding a cytochrome P450 family protein with trans-hydroxylation enzyme activity that forms trans-zeatin (tZ)-type cytokinins from N6(Δ2-isopentenyl) adenine (iP)-type cytokinins. This enzyme plays a crucial role in cytokinin biosynthesis and homeostasis in plants. The significance of CYP735A2 lies in its ability to modify side-chain structures of cytokinins, which directly affects their biological activity and transport mechanisms. Current research indicates that CYP735A2 is one of the major genes responsible for maintaining active cytokinin homeostasis, making it a valuable target for studies of plant development, stress responses, and hormone signaling pathways .

Where is CYP735A2 primarily expressed in plant tissues?

CYP735A2 is predominantly expressed in root tissues, with higher expression levels also documented during petal differentiation, in hypocotyls, and in the leaf-forming structures of the shoot apical meristem. The protein has been localized to multiple subcellular compartments, including mitochondria and extracellular regions. Notably, proteomic studies have identified the protein in plasmodesmata, suggesting potential roles in cell-to-cell communication . This tissue-specific and subcellular expression pattern is important to consider when designing antibody-based experiments to detect CYP735A2 in different plant organs and cellular fractions.

How does CYP735A2 expression respond to cytokinin treatment?

Research demonstrates that the CYP735A2 transcript is induced by all forms of active cytokinins, including the synthetic cytokinin benzyladenine (BA). This responsiveness distinguishes CYP735A2 from its paralog CYP735A1, which remains insensitive to cytokinin treatment. The CYP735A2 promoter contains several core motifs and one extended motif that bind type-B response regulators (RRs), establishing a direct link between this gene and the immediate-early cytokinin signaling network . This regulatory mechanism creates a feedback loop where cytokinins promote their own biosynthesis through increased CYP735A2 expression, which researchers should account for when interpreting antibody-based protein detection results following hormone treatments.

What are the optimal methods for generating specific antibodies against CYP735A2?

For generating high-quality CYP735A2-specific antibodies, researchers should consider a combined approach of both peptide and recombinant protein antigens. The methodology should mirror successful antibody production strategies used for related plant proteins. For instance, polyclonal antibodies can be prepared by immunizing rabbits with purified protein or synthetic peptides corresponding to unique regions of CYP735A2. The antibodies should be affinity-purified using column chromatography methods such as HiTrap Mab purification followed by epitope selection techniques to enhance specificity .

When designing immunogenic peptides, researchers should target unique regions of CYP735A2 that don't share sequence homology with CYP735A1 or other cytochrome P450 proteins. Epitope prediction software should be employed to identify hydrophilic, surface-exposed regions that are likely to be immunogenic. Validation should include western blotting against both native plant extracts and heterologously expressed CYP735A2 protein to confirm specificity and minimize cross-reactivity.

How can researchers effectively validate CYP735A2 antibody specificity?

Rigorous validation of CYP735A2 antibodies requires a multi-faceted approach:

• Western blot analysis comparing wild-type plants with cyp735a2 knockout/knockdown mutants

• Preabsorption tests with the immunizing peptide or recombinant protein

• Testing against recombinant CYP735A2 expressed in heterologous systems

• Cross-reactivity assessment with the paralog CYP735A1 and other related cytochrome P450 enzymes

• Immunoprecipitation followed by mass spectrometry to confirm target identity

Researchers should observe a single band of appropriate molecular weight (~55-60 kDa) for CYP735A2 in western blots that is absent or reduced in knockout/knockdown lines. Additionally, antibody signal should be blocked when preincubated with the immunizing antigen, confirming binding specificity . These validation steps are critical before proceeding with extensive experimental applications to ensure accurate interpretation of results.

What are the most effective techniques for determining CYP735A2 subcellular localization using antibodies?

The subcellular localization of CYP735A2 can be accurately determined using a combination of cell fractionation and immunodetection techniques. Based on methodologies used for similar proteins, researchers should:

• Isolate intact organelles (plastids, mitochondria, and other cellular compartments) using established differential centrifugation protocols

• Verify fraction purity using marker proteins specific to different organelles

• Perform western blot analysis with anti-CYP735A2 antibodies on each fraction

• Complement biochemical fractionation with immunofluorescence microscopy

For example, isolation of intact plastids from Arabidopsis seedlings can follow protocols similar to those used for studying other proteins (as described in the literature) . The presence of CYP735A2 in different fractions should be compared to established marker proteins such as ferredoxin (stroma), phosphoenolpyruvate carboxylase (cytosol), binding protein (endoplasmic reticulum), and mitochondrial aspartate aminotransferase .

How can organelle integrity be verified during subcellular localization studies of CYP735A2?

To ensure accurate subcellular localization data, researchers must verify the integrity of isolated organelles when studying CYP735A2 distribution. The recommended approach involves protease protection assays using proteases with different membrane penetration capabilities, such as trypsin and thermolysin.

For example, in studies of plastid-localized proteins, researchers have demonstrated that stromal proteins remain resistant to both trypsin and thermolysin treatments, while envelope proteins show differential sensitivity . When applying this methodology to CYP735A2 studies, researchers should:

• Expose isolated organelle fractions to trypsin or thermolysin under controlled conditions

• Compare the protease sensitivity pattern of CYP735A2 to known marker proteins

• Include detergent treatments (e.g., Triton X-100) as positive controls to destroy membrane barriers

• Analyze protein degradation patterns by western blotting with CYP735A2 antibodies

The resistance or sensitivity to these proteases provides crucial information about whether CYP735A2 is located within organelles or associated with membranes .

How can CYP735A2 antibodies be effectively used in western blot analyses of plant tissues?

For optimal western blot detection of CYP735A2 in plant tissues, researchers should follow this methodological approach:

• Homogenize approximately 400 mg of plant tissue in two volumes of ice-cold extraction buffer (50 mM Tris·HCl, 150 mM NaCl, pH 7.5)

• Clarify extracts by centrifugation and quantify protein concentration

• Separate proteins using SDS-PAGE (10-12% acrylamide gels are typically suitable)

• Transfer proteins to PVDF or nitrocellulose membranes

• Block membranes with 5% non-fat milk or BSA in TBST

• Incubate with primary anti-CYP735A2 antibodies (optimized dilution, typically 1:1000 to 1:5000)

• Apply appropriate secondary antibodies conjugated to HRP or fluorophores

• Develop using chemiluminescence or fluorescence detection systems

When analyzing CYP735A2 expression across different tissues, researchers should normalize loading using housekeeping proteins such as actin or GAPDH. For definitive identification, comparisons with cyp735a2 mutant tissues and recombinant protein standards are recommended . This approach will yield reliable data on CYP735A2 protein abundance across different experimental conditions.

What considerations are important when designing co-immunoprecipitation experiments with CYP735A2 antibodies?

When designing co-immunoprecipitation (co-IP) experiments to identify CYP735A2 interacting partners, researchers should consider several critical factors:

• Buffer composition: Use mild, non-denaturing buffers that preserve protein-protein interactions (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40 or 0.5% Triton X-100, 1 mM EDTA, with protease inhibitors)

• Crosslinking options: Consider reversible crosslinkers like DSP (dithiobis(succinimidyl propionate)) for transient interactions

• Antibody coupling: Covalently couple purified CYP735A2 antibodies to protein A/G beads or magnetic beads to prevent antibody contamination in mass spectrometry analysis

• Controls: Include IgG from the same species as negative controls and input samples for comparison

• Validation: Confirm interactions through reciprocal co-IPs and alternative methods like yeast two-hybrid or BiFC

The co-IP workflow should involve tissue extraction, pre-clearing with protein A/G beads, incubation with antibody-coupled beads, stringent washing, and elution followed by immunoblotting or mass spectrometry . This approach can reveal previously unknown protein interactions that may provide insight into CYP735A2 regulation and function in cytokinin biosynthesis pathways.

How can CYP735A2 antibodies be used to investigate enzyme regulation during plant stress responses?

CYP735A2 antibodies provide a powerful tool for investigating changes in enzyme abundance and post-translational modifications during plant stress responses. To effectively study these changes, researchers should employ a comprehensive approach:

• Subject plants to well-defined stress treatments (drought, salt, temperature, pathogen infection)

• Harvest tissue samples at multiple time points following stress application

• Perform western blot analysis using CYP735A2 antibodies to track protein abundance

• Combine with RT-qPCR to correlate protein levels with transcript abundance

• Investigate post-translational modifications using phospho-specific antibodies or mass spectrometry following immunoprecipitation

For phosphorylation studies, researchers should immunoprecipitate CYP735A2 from stressed and control tissues, then analyze samples using phospho-specific staining or mass spectrometry. Given the known inhibition of CYP735A enzymes by uniconazole , researchers should also consider how stress-induced changes in CYP735A2 activity might be regulated by small molecule inhibitors or protein-protein interactions that could be detected using antibody-based approaches.

What methodological approaches enable the investigation of CYP735A2 protein-protein interactions in planta?

To investigate CYP735A2 protein-protein interactions in planta, researchers should implement multiple complementary approaches:

• Proximity-dependent biotin labeling (BioID or TurboID):

  • Generate transgenic plants expressing CYP735A2 fused to a biotin ligase

  • Induce biotinylation of proteins in proximity to CYP735A2

  • Purify biotinylated proteins using streptavidin

  • Identify interaction partners by mass spectrometry

• Immunoprecipitation coupled with crosslinking:

  • Apply membrane-permeable crosslinkers to intact plant tissues

  • Immunoprecipitate CYP735A2 using specific antibodies

  • Identify crosslinked proteins by mass spectrometry

• Split-reporter protein complementation with antibody validation:

  • Generate split-YFP/GFP/luciferase fusions with CYP735A2 and candidate interactors

  • Visualize interactions in planta

  • Validate using co-immunoprecipitation with CYP735A2 antibodies

Each identified interaction should be validated using multiple approaches and assessed for biological relevance in cytokinin biosynthesis pathways. The combination of these techniques can provide a comprehensive interactome for CYP735A2, revealing new insights into how this enzyme is regulated in different developmental contexts .

How can researchers address non-specific binding issues when using CYP735A2 antibodies?

Non-specific binding is a common challenge when working with antibodies against plant proteins like CYP735A2. To address this issue, researchers should implement the following optimization strategies:

• Blocking optimization:

  • Test different blocking agents (5% non-fat milk, 3-5% BSA, commercial blocking buffers)

  • Extend blocking time to 2-3 hours at room temperature or overnight at 4°C

• Antibody dilution optimization:

  • Perform titration experiments with serial dilutions (1:500 to 1:10,000)

  • Determine optimal concentration that maximizes specific signal while minimizing background

• Stringent washing:

  • Increase washing duration and frequency (5-6 washes of 10 minutes each)

  • Add low concentrations of detergents (0.1-0.3% Tween-20 or 0.05% SDS) to washing buffers

• Pre-adsorption:

  • Incubate antibodies with proteins extracted from cyp735a2 knockout plants

  • Remove antibodies that bind to non-specific targets before using with experimental samples

These optimizations should be systematically tested and documented to establish a reliable protocol that produces consistent, specific detection of CYP735A2 across different tissue types and experimental conditions .

What are the most effective extraction methods for maintaining CYP735A2 integrity during immunodetection?

Preserving CYP735A2 protein integrity during extraction is critical for accurate immunodetection. Based on protocols used for similar cytochrome P450 proteins, researchers should consider these methodological approaches:

• Buffer composition:

  • Use 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1 mM EDTA

  • Add protease inhibitor cocktail (e.g., PMSF, leupeptin, aprotinin)

  • Include reducing agents (5 mM DTT or 2 mM β-mercaptoethanol) to maintain protein structure

• Extraction conditions:

  • Maintain samples at 4°C throughout processing

  • Use gentle homogenization methods to prevent protein denaturation

  • Consider nitrogen grinding for recalcitrant tissues

• Membrane protein considerations:

  • Add mild detergents (0.5-1% Triton X-100, 0.5% NP-40, or 0.1% digitonin) to solubilize membrane-associated CYP735A2

  • Centrifuge at different speeds to separate cellular fractions if studying subcellular distribution

• Sample preparation for electrophoresis:

  • Avoid extended boiling in SDS sample buffer (limit to 5 minutes at 95°C)

  • Consider alternative sample denaturation at 70°C for 10 minutes for membrane proteins

These optimized extraction protocols will ensure maximum recovery of intact CYP735A2 protein, enhancing detection sensitivity and experimental reproducibility .

How should researchers quantify CYP735A2 protein levels across different developmental stages?

For accurate quantification of CYP735A2 protein levels across developmental stages, researchers should implement a rigorous analytical approach:

• Standardized sample preparation:

  • Collect tissues at precisely defined developmental stages

  • Process all samples simultaneously to minimize batch effects

  • Include multiple biological replicates (minimum n=3)

• Quantitative western blot analysis:

  • Include recombinant CYP735A2 protein standards at known concentrations

  • Apply samples in technical triplicates

  • Use fluorescent secondary antibodies for wider linear dynamic range

• Normalization strategy:

  • Normalize against multiple housekeeping proteins (actin, GAPDH, tubulin)

  • Consider total protein normalization using stain-free gels or Ponceau S staining

  • Verify stability of reference proteins across developmental stages

• Statistical analysis:

  • Apply appropriate statistical tests for developmental time course data

  • Calculate coefficient of variation between replicates (aim for CV < 15%)

  • Report both absolute and relative quantification values

A representative data table for CYP735A2 quantification across developmental stages should include columns for developmental stage, biological replicate number, raw CYP735A2 signal intensity, normalization factor, normalized CYP735A2 levels, and statistical significance indicators .

How can researchers correlate CYP735A2 protein abundance with cytokinin levels in plant tissues?

To establish meaningful correlations between CYP735A2 protein abundance and cytokinin levels, researchers should implement this integrated analytical workflow:

• Parallel sample processing:

  • Divide each tissue sample into two portions

  • Process one portion for CYP735A2 immunodetection

  • Extract cytokinins from the other portion using established protocols

• Cytokinin profiling:

  • Quantify multiple cytokinin species (especially iP- and tZ-type cytokinins)

  • Use liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Include stable isotope-labeled internal standards

• Correlation analysis:

  • Calculate Pearson or Spearman correlation coefficients between CYP735A2 abundance and individual cytokinin species

  • Perform multivariate analysis to identify patterns across cytokinin profiles

  • Create scatter plots with regression lines to visualize relationships

• Validation experiments:

  • Manipulate CYP735A2 expression using inducible expression systems

  • Monitor resulting changes in cytokinin profiles

  • Establish cause-effect relationships through time-course analyses

This integrated approach permits researchers to determine whether CYP735A2 protein abundance is a reliable predictor of specific cytokinin species accumulation, particularly the ratio between iP- and tZ-type cytokinins, which is a critical aspect of CYP735A2 function .