AOP3 Antibody

What is AOP3 protein and why is it studied in plant research?

AOP3 (ALKENYL HYDROXALKYL PRODUCING 3) is a protein expressed in Arabidopsis thaliana with Uniprot accession number Q9ZTA1 . It functions in glucosinolate biosynthesis pathways, which are critical for plant defense against herbivores and pathogens. Research on AOP3 helps understand plant biochemical defense mechanisms, metabolic regulation, and evolutionary adaptations to environmental stressors. Studies typically employ AOP3 antibodies to track protein expression, localization, and interactions within plant tissues.

What types of AOP3 antibodies are available for research?

AOP3 antibodies are primarily available as polyclonal antibodies raised in rabbits against recombinant Arabidopsis thaliana AOP3 protein . These antibodies are supplied in liquid form with storage buffers containing glycerol (typically 50%) and preservatives like Proclin 300 (0.03%) . While monoclonal options exist for many plant proteins, polyclonal antibodies often provide advantages in plant research due to their ability to recognize multiple epitopes on the target protein, enhancing detection sensitivity in complex plant tissue samples.

What applications are validated for AOP3 antibodies?

AOP3 antibodies have been validated primarily for Western blotting (WB) and Enzyme-Linked Immunosorbent Assay (ELISA) applications . These techniques enable researchers to:

While not explicitly validated in the product documentation, experienced researchers may adapt these antibodies for immunoprecipitation, immunohistochemistry, or immunofluorescence microscopy after appropriate optimization and validation steps.

How should I design optimal Western blot experiments with AOP3 antibody?

When designing Western blot experiments to detect AOP3 protein in Arabidopsis samples, consider the following methodological approach:

• Sample preparation: Extract proteins using a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, and protease inhibitor cocktail. Plant tissues should be flash-frozen and ground in liquid nitrogen before extraction.

• Protein separation: Use 10-12% SDS-PAGE gels for optimal resolution of AOP3 (check expected molecular weight from UniProt data).

• Transfer conditions: For plant proteins like AOP3, semi-dry transfer at 15V for 30-45 minutes using PVDF membranes typically yields better results than nitrocellulose.

• Blocking: Block membranes with 5% non-fat dry milk in TBST (TBS with 0.1% Tween-20) for 1 hour at room temperature.

• Primary antibody incubation: Dilute AOP3 antibody to 2 μg/ml in blocking buffer and incubate overnight at 4°C with gentle rocking .

• Secondary antibody: Use an alkaline phosphatase-labeled goat anti-rabbit antibody at approximately 160 ng/ml concentration .

• Detection: For optimal sensitivity with minimal background, use NBT/BCIP substrate diluted 1:100 in alkaline phosphatase buffer (100 mM Tris-HCl pH 9.6, 100 mM NaCl, 5 mM MgCl₂) .

• Controls: Always include both positive controls (wild-type Arabidopsis extracts) and negative controls (AOP3 knockout mutants if available) to verify antibody specificity.

What are the recommended storage conditions for maintaining AOP3 antibody activity?

To maintain optimal activity of AOP3 antibody:

• Upon receipt, immediately aliquot the antibody into small volumes (10-20 μl) to minimize freeze-thaw cycles.

• Store at -20°C or preferably -80°C for long-term storage .

• Avoid repeated freeze-thaw cycles as this can lead to antibody denaturation and reduced activity.

• For working stocks, store small aliquots at 4°C with 0.02% sodium azide for up to 2 weeks.

• Before each use, centrifuge the antibody vial briefly to collect contents at the bottom of the tube.

• Transport on ice when moving between storage and experimental areas.

Proper storage significantly impacts experimental reproducibility, as improperly stored antibodies may show diminished sensitivity or increased background in detection assays.

How can I troubleshoot weak or absent signals when using AOP3 antibody?

When facing weak or absent signals in AOP3 detection experiments, systematically address these potential issues:

Remember that plant tissues contain many compounds that can interfere with antibody-based detection, including phenolics, tannins, and polysaccharides. Adding polyvinylpyrrolidone (PVP) to extraction buffers can help mitigate these interferences.

How can I validate the specificity of AOP3 antibody for my experiments?

Validating antibody specificity is crucial for generating reliable research data. For AOP3 antibody, implement these validation approaches:

• Genetic controls: Test the antibody on tissues from AOP3 knockout or knockdown Arabidopsis lines, which should show reduced or absent signal.

• Blocking peptide competition: Pre-incubate the antibody with purified recombinant AOP3 protein before application to your samples; specific binding should be significantly reduced.

• Multiple detection methods: Confirm findings using multiple techniques (e.g., if detected by Western blot, also confirm with ELISA) .

• Molecular weight verification: Ensure the detected protein matches the expected molecular weight of AOP3 in Arabidopsis.

• 2D gel electrophoresis: For advanced validation, use 2D gel electrophoresis followed by immunoblotting to confirm specificity based on both molecular weight and isoelectric point .

• Mass spectrometry confirmation: After immunoprecipitation with AOP3 antibody, subject the captured proteins to mass spectrometry analysis to confirm target identity.

How can AOP3 antibody be used to investigate protein-protein interactions?

To investigate protein-protein interactions involving AOP3:

• Co-immunoprecipitation (Co-IP): Use AOP3 antibody conjugated to protein A/G beads to pull down AOP3 and its interacting partners from plant lysates. Analyze the precipitated complexes by mass spectrometry or Western blotting with antibodies against suspected interaction partners.

• Proximity ligation assay (PLA): Combine AOP3 antibody with antibodies against potential interaction partners to visualize protein interactions in situ with sub-cellular resolution.

• FRET-based approaches: For advanced applications, combine immunofluorescence using AOP3 antibody with fluorescently-tagged candidate interacting proteins.

• Yeast two-hybrid confirmation: Use AOP3 antibody to validate interactions initially discovered through yeast two-hybrid screens by confirming co-localization in plant tissues.

When designing these experiments, consider including appropriate controls such as non-specific IgG for Co-IP and single-antibody controls for PLA to rule out false positives.

What are the considerations for using AOP3 antibody in comparative studies across different plant species?

When extending AOP3 research beyond Arabidopsis thaliana to other plant species:

• Sequence homology assessment: Analyze the sequence conservation of AOP3 epitopes between Arabidopsis and your target species. Higher similarity increases the likelihood of cross-reactivity.

• Validation in target species: Perform Western blot analysis on protein extracts from the new species to confirm antibody cross-reactivity and determine optimal working conditions.

• Tissue-specific expression: Consider that AOP3 expression patterns may differ substantially between species, requiring adjustment of sampling strategies and protein extraction protocols.

• Epitope accessibility: Post-translational modifications or protein folding differences might affect epitope recognition in different species.

• Control samples: Include Arabidopsis samples as positive controls alongside your experimental species samples in all experiments.

Cross-species applications often require higher antibody concentrations and longer incubation times to achieve comparable results to those obtained in Arabidopsis.

How can I quantify AOP3 expression levels using antibody-based techniques?

For accurate quantification of AOP3 protein levels:

• ELISA-based quantification:

  • Develop a standard curve using purified recombinant AOP3 protein

  • Use indirect ELISA with AOP3 antibody as the primary antibody

  • Calculate protein concentration from the standard curve

• Western blot densitometry:

  • Include a dilution series of recombinant AOP3 protein on each blot

  • Use housekeeping proteins (e.g., actin or tubulin) as loading controls

  • Analyze band intensities using ImageJ or similar software

  • Ensure signal is within the linear range of detection

• Data normalization methods:

  • For ELISA: Normalize to total protein concentration determined by Bradford or BCA assay

  • For Western blot: Express as ratio to housekeeping protein or as percentage of control samples

• Statistical analysis:

  • Perform at least three biological replicates

  • Use appropriate statistical tests (t-test for two-condition comparisons; ANOVA for multiple conditions)

  • Report results with standard deviation or standard error

What factors might influence antigen-antibody interactions when using AOP3 antibody?

Several factors can impact the interaction between AOP3 antibody and its target antigen:

Understanding these factors allows researchers to optimize experimental conditions for both qualitative and quantitative applications involving AOP3 antibody.

How is AOP3 antibody being used in current plant immunity and stress response research?

Current research applications for AOP3 antibody in plant immunity studies include:

• Monitoring AOP3 protein levels in response to pathogen challenge to understand its role in the glucosinolate-mediated defense pathway.

• Investigating post-translational modifications of AOP3 during various stress conditions using phospho-specific antibodies in conjunction with general AOP3 antibodies.

• Examining tissue-specific and subcellular localization patterns of AOP3 under different environmental stresses.

• Studying AOP3 protein turnover rates during plant development and stress responses using pulse-chase experiments combined with immunoprecipitation.

• Investigating potential conservation of AOP3-mediated responses across different plant species as part of comparative immunology studies.

These applications help researchers understand the molecular mechanisms underlying plant defense responses and potentially identify targets for improving crop resistance to biotic and abiotic stresses.

What are the challenges in generating autoantibody profiles that might cross-react with plant proteins like AOP3?

Research into autoantibody profiles faces several challenges when investigating potential cross-reactivity with plant proteins:

• Molecular mimicry: Short peptide sequences in plant proteins like AOP3 may share similarity with human proteins, potentially triggering cross-reactivity . Studies show that 7-8 amino acid ungapped matches between proteins can be sufficient for antibody cross-reactivity .

• Age-dependent variations: Autoantibody profiles change with age, with numbers increasing during childhood and adolescence before plateauing . This developmental pattern must be considered when studying potential plant protein cross-reactivity.

• Biochemical properties affecting immunogenicity: Common autoantigens often share properties like low aromaticity, high hydrophilicity, and flexibility . Analysis of AOP3's biochemical properties would be necessary to predict its potential cross-reactivity with human autoantibodies.

• Detection limitations: Technical challenges in distinguishing specific from non-specific binding can complicate cross-reactivity studies, requiring careful experimental design with appropriate controls.

Understanding these challenges is essential when using AOP3 antibody in research that may have implications for human health, such as studies investigating plant-based dietary triggers of autoimmune responses.