DHAR2 Antibody

What is DHAR2 and why are antibodies against it important in plant research?

DHAR2 (Dehydroascorbate Reductase 2) is a critical enzyme in plants involved in the ascorbate-glutathione cycle, which plays an essential role in antioxidant defense systems. In Arabidopsis thaliana, DHAR2 (At1g75270) helps maintain redox homeostasis by catalyzing the reduction of dehydroascorbate to ascorbate using glutathione as a reducing agent.

Antibodies against DHAR2, such as polyclonal antibodies raised in rabbits using KLH-conjugated synthetic peptides derived from the DHAR sequence, are crucial research tools that enable:

• Monitoring DHAR2 expression levels under various stress conditions

• Determining subcellular localization through immunohistochemistry

• Studying protein-protein interactions via co-immunoprecipitation

• Evaluating post-translational modifications

Researchers targeting plant stress physiology, redox biology, and antioxidant systems frequently employ these antibodies to advance understanding of plant responses to environmental stressors .

How can I verify the specificity of my DHAR2 antibody?

Verifying antibody specificity is a critical step before conducting extensive experiments. For DHAR2 antibodies, implement the following validation protocol:

• Western blot with positive and negative controls:

  • Positive control: Recombinant DHAR2 protein or extract from wild-type plants

  • Negative control: Extract from DHAR2 knockout/knockdown plants

• Cross-reactivity assessment:

  • Test against purified related proteins (DHAR1, DHAR3)

  • Compare banding patterns with predicted molecular weights

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide

  • Signal should be significantly reduced in Western blot

• Immunoprecipitation followed by mass spectrometry:

  • Confirm pulled-down protein is indeed DHAR2

• Correlation with transcript levels:

  • Compare protein detection with RT-qPCR results in different conditions

This multi-approach validation ensures that experimental observations are truly attributable to DHAR2 and not to cross-reactivity with related proteins .

What are the optimal conditions for using DHAR2 antibodies in Western blotting?

Optimized Western blot protocol for DHAR2 antibody detection:

Sample Preparation:

• Extract proteins in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA with freshly added protease inhibitors

• For plant tissues, add 10 mM DTT and 1% PVPP to reduce interference from phenolic compounds

Gel Electrophoresis and Transfer:

• Use 12-15% SDS-PAGE gels (DHAR2 is approximately 23-25 kDa)

• Transfer to PVDF membrane at 100V for 60 minutes in 10% methanol transfer buffer

Antibody Incubation:

• Blocking: 5% non-fat dry milk in TBS-T (1 hour at room temperature)

• Primary antibody: Dilute DHAR2 antibody 1:1000 to 1:2000 in 1% BSA/TBS-T

• Incubation: Overnight at 4°C with gentle rocking

• Secondary antibody: Anti-rabbit HRP at 1:5000 dilution (1 hour at room temperature)

Detection and Troubleshooting:

• Use ECL substrate for standard detection

• Expected band: ~23-25 kDa for Arabidopsis DHAR2

• If background is high, increase washing time and reduce primary antibody concentration

• For weak signals, extend primary antibody incubation and use high-sensitivity ECL substrate

This protocol has been validated across multiple plant species and tissue types with consistent results .

How can I optimize immunolocalization studies using DHAR2 antibodies?

Successful immunolocalization of DHAR2 in plant tissues requires careful protocol optimization:

Tissue Preparation:

• Fix tissue in 4% paraformaldehyde in PBS (pH 7.4) for 2-4 hours

• Embedded in paraffin or resin depending on required resolution

• For whole-mount preparations, use 2% paraformaldehyde with 0.1% glutaraldehyde

Antigen Retrieval:

• Perform heat-induced epitope retrieval in 10 mM sodium citrate buffer (pH 6.0)

• Alternative: Enzymatic retrieval with 0.01% trypsin in PBS for 10-15 minutes at 37°C

Immunostaining Protocol:

• Blocking: 5% normal goat serum, 1% BSA in PBS with 0.1% Triton X-100 (2 hours)

• Primary antibody: Dilute DHAR2 antibody 1:100 to 1:200 in blocking buffer

• Incubation: 12-16 hours at 4°C in a humid chamber

• Secondary antibody: Fluorophore-conjugated anti-rabbit IgG at 1:300 dilution

Controls and Visualization:

• Negative controls: Pre-immune serum and secondary antibody-only samples

• For co-localization studies, pair with established organelle markers:

  • Chloroplast: anti-RbcL

  • Peroxisome: anti-catalase

  • Cytosol: anti-GAPDH (cytosolic isoform)

Results Interpretation:

• DHAR2 typically shows cytosolic and chloroplastic localization patterns

• Confirm specificity by comparing with subcellular fractionation results and GFP fusion studies

This optimized protocol minimizes background staining while maximizing specific DHAR2 detection .

What are the common causes of inconsistent results with DHAR2 antibodies and how can they be addressed?

Each troubleshooting approach should be systematically documented to identify the specific variables affecting your experimental system. Creating standardized protocols with internal controls significantly improves reproducibility when working with DHAR2 antibodies across different plant stress conditions and developmental stages .

How should I interpret contradictory results between DHAR2 protein levels and enzyme activity measurements?

When DHAR2 protein levels detected by antibodies contradict enzyme activity measurements, consider these potential explanations and resolution strategies:

Potential Causes of Discrepancies:

• Post-translational Modifications

  • Phosphorylation, glutathionylation, or oxidation may alter DHAR2 activity without changing protein abundance

  • Resolution: Combine Western blots with Phos-tag gels or redox proteomics techniques

• Protein-Protein Interactions

  • DHAR2 activity can be regulated by protein partners not detectable in standard assays

  • Resolution: Perform co-immunoprecipitation followed by activity assays of the complex

• Substrate/Cofactor Availability

  • In vivo glutathione limitations may restrict activity despite high protein levels

  • Resolution: Measure GSH/GSSG ratios in corresponding samples

• Compartmentalization Effects

  • DHAR2 may be sequestered in different cellular compartments affecting its activity

  • Resolution: Combine immunolocalization with subcellular fractionation and activity assays

Methodological Approach for Resolution:

• Perform parallel analysis of DHAR2 protein levels (Western blot), transcript abundance (RT-qPCR), and enzyme activity under identical conditions

• Assess protein turnover rates using cycloheximide chase experiments

• Evaluate the redox state of the cellular environment

• Consider isoform-specific activities if multiple DHAR enzymes are present

This comprehensive analysis approach helps determine whether discrepancies represent technical artifacts or genuine biological regulation mechanisms, such as post-translational activity control .

How can DHAR2 antibodies be utilized in mass spectrometry-based proteomics studies?

DHAR2 antibodies can significantly enhance mass spectrometry-based proteomics studies through several advanced applications:

Immunoprecipitation-Mass Spectrometry (IP-MS):

• Use DHAR2 antibodies conjugated to magnetic beads or protein A/G

• Enrich DHAR2 and associated proteins from complex cellular extracts

• Perform on-bead digestion with trypsin before LC-MS/MS analysis

• Identify DHAR2 interaction partners under various stress conditions

Selected Reaction Monitoring (SRM) Enhancement:

• Develop SRM assays targeting DHAR2-specific peptides identified through antibody-based enrichment

• Create a spectral library of DHAR2 peptides for accurate quantification

• Enhance detection sensitivity by 10-100 fold through preliminary immunoenrichment

Post-Translational Modification Mapping:

• Enrich DHAR2 using specific antibodies

• Analyze enriched fractions using LC-MS/MS with neutral loss scanning for phosphorylation

• Apply electron transfer dissociation (ETD) for precise localization of modifications

Workflow Implementation:

• Optimize DHAR2 antibody binding conditions for maximum specificity

• Perform parallel immunoprecipitations with specific antibody and non-specific IgG control

• Analyze eluates using high-resolution MS (Orbitrap or Q-TOF)

• Apply SAINT or similar statistical models to identify high-confidence interactors

• Validate key interactions through reciprocal co-immunoprecipitation

This approach has revealed that DHAR2 interacts with components of the ascorbate-glutathione cycle and stress-responsive signaling proteins, providing deeper insights into redox signaling networks in plants .

What approaches can be used to develop machine learning models for DHAR2-related disease detection using antibody-derived peptides?

Developing machine learning models for DHAR2-related disease detection using antibody-derived peptides involves several sophisticated approaches:

Data Acquisition and Preparation:

• Compile DHAR2 antibody sequence datasets from sources like OAS (Observed Antibody Space)

• Perform in silico digestion to generate theoretical peptide fragments

• Remove peptides present in common databases to identify unique signature peptides

• Create databases of varying sizes (10² to 10⁷ peptides) to optimize search space and minimize false discovery rates

Mass Spectrometry Data Processing:

• Process MS data using database search algorithms (e.g., Mascot, SEQUEST)

• Apply target-decoy approach to control false discovery rate

• Implement spectral matching techniques for peptide identification

• Validate identifications using synthetic peptide standards

Machine Learning Model Development:

• Feature extraction from MS/MS spectra and antibody sequence patterns

• Apply dimensionality reduction techniques (PCA, t-SNE)

• Test multiple algorithms:

  • Support Vector Machines

  • Random Forests

  • Deep Neural Networks (particularly CNN for spectral data)

• Implement k-fold cross-validation to assess model performance

Validation and Performance Metrics:

• Sensitivity and specificity in distinguishing disease states

• Area Under ROC Curve (AUC) for model comparison

• Positive Predictive Value for clinical relevance assessment

Example Performance Comparison:

This approach leverages the unique peptide signatures derived from DHAR2 antibodies to develop diagnostic tools with potential applications in plant stress detection, offering a promising avenue for translating fundamental research into practical biotechnology applications .

How can I design experiments to investigate DHAR2 antibody cross-reactivity with other DHAR family members?

A systematic approach to investigate cross-reactivity between DHAR2 antibodies and other DHAR family members requires careful experimental design:

Step 1: Sequence Analysis and Epitope Mapping

• Perform multiple sequence alignment of all DHAR family proteins

• Identify regions of high similarity that may cause cross-reactivity

• Map known epitopes recognized by the antibody

• Use computational tools to predict potential cross-reactive epitopes

Step 2: Recombinant Protein Expression

• Clone and express all DHAR family members (DHAR1, DHAR2, DHAR3) with appropriate tags

• Purify proteins under native conditions to maintain structural integrity

• Quantify protein concentration accurately using BCA or Bradford assays

Step 3: Cross-Reactivity Assessment Protocol

• ELISA-based quantification:

  • Coat plates with equal amounts (100 ng/well) of each DHAR protein

  • Test DHAR2 antibody at multiple dilutions (1:500 to 1:10,000)

  • Calculate relative affinity for each protein

• Western blot analysis:

  • Load equivalent amounts (25-50 ng) of each recombinant protein

  • Perform Western blotting with standardized conditions

  • Quantify band intensity using densitometry

  • Calculate cross-reactivity ratios

• Surface Plasmon Resonance (SPR):

  • Immobilize DHAR2 antibody on sensor chip

  • Flow solutions containing each DHAR protein

  • Determine binding kinetics (kon, koff) and affinity constants (KD)

Step 4: Validation in Biological Samples

• Analyze extracts from wild-type plants and single/multiple DHAR knockout lines

• Perform immunoprecipitation followed by mass spectrometry to identify pulled-down proteins

• Use immunofluorescence in plant tissues with known differential expression of DHAR isoforms

This comprehensive approach provides quantitative data on cross-reactivity potential, enabling researchers to correctly interpret experimental results and design appropriate controls for DHAR2-specific studies .

What experimental designs can distinguish between DHAR2 protein abundance changes and post-translational modifications?

Distinguishing between changes in DHAR2 protein abundance and post-translational modifications requires a multi-faceted experimental approach:

Comprehensive Experimental Design:

• Parallel Western Blot Analysis:

  • Standard Western blot for total DHAR2 protein

  • Phos-tag™ PAGE for phosphorylated forms

  • Non-reducing gels for detection of S-glutathionylation

  • 2D gels (IEF/SDS-PAGE) to separate modified isoforms

• Mass Spectrometry-Based Approaches:

  • DHAR2 immunoprecipitation followed by tryptic digestion

  • Parallel analysis with:

    • Standard peptide detection for abundance

    • Neutral loss scanning for phosphorylation

    • Precursor ion scanning for glutathionylation

    • MS^3 for complex modifications

• Targeted Post-Translational Modification Detection:

  • Phosphorylation: Use phospho-specific antibodies if available

  • Redox modifications: OxiRAC methodology to detect cysteine oxidation

  • Ubiquitination: Use anti-ubiquitin antibodies following DHAR2 immunoprecipitation

• Activity Correlation Studies:

  • Measure DHAR2 enzyme activity in parallel samples

  • Correlate changes in activity with specific modifications

  • Use site-directed mutagenesis of key residues to confirm functional impact

Data Integration Framework:

This integrated approach enables researchers to determine whether changes in DHAR2 function result from altered protein abundance or from specific post-translational modifications that affect activity, localization, or interaction with partner proteins .

How can DHAR2 antibodies be utilized in investigating plant responses to emerging climate stressors?

DHAR2 antibodies provide powerful tools for investigating plant responses to emerging climate stressors through various advanced research approaches:

Temporal and Spatial Profiling of Stress Responses:

• Use DHAR2 antibodies to track protein expression patterns during:

  • Extreme temperature fluctuations

  • Drought-flood cycles

  • Elevated CO₂ conditions

  • Combined stresses that mimic future climate scenarios

• Implement high-throughput immunoblotting across tissue types and developmental stages

Stress Priming and Memory Studies:

• Track DHAR2 protein levels during:

  • Initial stress exposure

  • Recovery phase

  • Subsequent stress challenge

• Correlate protein abundance with ascorbate redox state and ROS signatures

• Investigate epigenetic modifications affecting DHAR2 expression during recurrent stress

Field-to-Lab Translation:

• Collect field samples from plants experiencing natural climate extremes

• Preserve samples with specialized fixatives that maintain protein modifications

• Analyze DHAR2 expression and modification patterns using antibody-based techniques

• Correlate findings with controlled laboratory experiments

Multi-Omics Integration Framework:

• Combine antibody-detected DHAR2 protein data with:

  • Transcriptomics (RNA-seq)

  • Metabolomics (ascorbate/dehydroascorbate ratios)

  • Physiological measurements (photosynthetic efficiency)

  • Growth parameters (biomass accumulation)

This integrated approach provides a comprehensive understanding of how redox homeostasis mechanisms respond to complex climate stress scenarios, potentially identifying DHAR2 regulatory patterns as biomarkers for stress resilience in crop improvement programs .

What methodologies can be applied to develop DHAR2 antibody arrays for high-throughput phenotyping?

Developing DHAR2 antibody arrays for high-throughput phenotyping requires innovative methodological approaches that combine immunological techniques with advanced detection systems:

Array Development Strategy:

• Antibody Immobilization Options:

  • Nitrocellulose-coated glass slides for standard arrays

  • 3D hydrogel surfaces for enhanced binding capacity

  • Microfluidic channels for dynamic interaction studies

  • Quantum dot-conjugated surfaces for enhanced sensitivity

• Multiplex Design Considerations:

  • Include antibodies against:

    • Total DHAR2 protein

    • Phosphorylated DHAR2 (multiple sites)

    • Other ascorbate-glutathione cycle enzymes

    • Stress-responsive marker proteins

  • Incorporate internal normalization controls

• Sample Preparation Protocol:

  • High-throughput protein extraction using robotics

  • Standardized buffer system optimized for maintaining protein modifications

  • Fluorescent labeling for detection

  • Optional: fractionate samples to reduce complexity

• Data Acquisition and Analysis Pipeline:

  • Automated image capture using high-resolution scanners

  • Signal quantification with dedicated software

  • Statistical analysis for large-scale comparisons

  • Machine learning algorithms for pattern recognition

Application Workflow for Plant Phenotyping:

This high-throughput approach enables:

• Screening hundreds of genotypes for DHAR2-related stress responses

• Temporal analysis of protein abundance and modifications

• Identification of superior genotypes with enhanced redox homeostasis

• Discovery of novel regulatory patterns in antioxidant networks

The resulting phenotypic data can directly inform breeding programs targeting climate resilience through enhanced antioxidant capacity .

What are the emerging trends in DHAR2 antibody applications for plant stress biology?

The application of DHAR2 antibodies in plant stress biology is evolving rapidly, with several emerging trends shaping future research directions:

Single-Cell Immunodetection:

• Adaptation of flow cytometry techniques for plant protoplasts

• Development of in situ proximity ligation assays for detecting DHAR2 interactions at cellular resolution

• Integration with microfluidic plant-on-chip platforms for real-time monitoring

Antibody Engineering for Enhanced Specificity:

• Development of recombinant antibody fragments (scFv, Fab) against DHAR2

• CRISPR-based epitope tagging for enhanced detection specificity

• Nanobody development for improved penetration into plant tissues

Integrative Multi-Stress Analysis:

• High-throughput immunoassays for screening DHAR2 responses across stress combinations

• Correlation of DHAR2 dynamics with global redox state markers

• Network analysis integrating DHAR2 with other stress response pathways

Translational Applications:

• Development of field-deployable immunochromatographic strips for rapid DHAR2 quantification

• Establishment of DHAR2 protein levels as biomarkers for stress resistance breeding

• Application in crop improvement programs targeting climate resilience

These emerging approaches are enabling researchers to move beyond traditional laboratory studies toward systems-level understanding of antioxidant defense mechanisms and their contribution to plant survival under increasingly complex environmental stressors .

How might advances in antibody research methodologies influence future studies of DHAR2 in plant systems?

Advances in antibody research methodologies are poised to transform future studies of DHAR2 in plant systems through several innovative approaches:

Technological Innovations and Their Applications:

• Synthetic Antibody Development:

  • Phage display libraries specifically designed for plant protein targets

  • In silico antibody design based on DHAR2 epitope mapping

  • Non-animal-derived recombinant antibodies with enhanced specificity

• Spatiotemporal Detection Systems:

  • Antibody-based biosensors for real-time tracking of DHAR2 in living plants

  • Optogenetic reporter systems linked to antibody binding events

  • Super-resolution microscopy with specially modified antibodies

• Multiplexed Analysis Platforms:

  • Digital spatial profiling combining DHAR2 antibodies with transcriptomic markers

  • Mass cytometry (CyTOF) adapted for plant cells with metal-labeled antibodies

  • Single-cell proteomics with antibody-based enrichment

• Computational Integration:

  • Machine learning algorithms for antibody epitope prediction

  • Molecular dynamics simulations of antibody-DHAR2 interactions

  • Network analysis integrating antibody-detected protein changes with metabolic pathways

Impact on Scientific Understanding:

These methodological advances will enable researchers to:

• Detect previously unobservable post-translational modifications

• Determine DHAR2 protein half-life and turnover rates during stress

• Identify cell-specific variations in DHAR2 function

• Discover novel interactions between DHAR2 and signaling pathways

• Establish causal relationships between DHAR2 activity and stress tolerance

Future Research Framework: