DHAR1 Antibody

What is DHAR1 and what is its biological significance?

DHAR1 (Dehydroascorbate Reductase 1) is a key component of the ascorbate recycling system involved in redox homeostasis under biotic and abiotic stress conditions. The protein is induced by jasmonic acid and oxidative chemical stresses and plays a crucial role in maintaining cellular redox balance. It is primarily localized in mitochondria and functions in glutathione-dependent reactions. DHAR1 is also known as glutathione-dependent dehydroascorbate reductase 1, chloride intracellular channel homolog 1 (CLIC homolog 1), AtDHAR1, and GSH-dependent dehydroascorbate reductase 1 (mtDHAR, AT1G19570) .

What are the molecular characteristics of DHAR1 protein that researchers should consider when selecting antibodies?

When selecting DHAR1 antibodies, researchers should consider that the protein has an expected molecular weight of approximately 23.6 kDa, though the apparent molecular weight on SDS-PAGE may be around 23.4 kDa. The protein structure contains specific epitopes that may be conserved across species such as Arabidopsis thaliana and Solanum lycopersicum, allowing for cross-reactivity in some cases. When designing experiments, consider that DHAR1 is part of a family of DHAR proteins with potentially similar sequences, necessitating careful antibody selection to avoid cross-reactivity with other family members like DHAR2 .

How do DHAR1 antibodies from different host species compare in terms of reactivity and applications?

DHAR1 antibodies are available with reactivity against various species, including Arabidopsis thaliana, Solanum lycopersicum (tomato), and Oryza sativa (rice). Most commonly, these are polyclonal antibodies raised in rabbits. The table below summarizes the characteristics of DHAR1 antibodies from different sources:

Different antibodies may show varying degrees of specificity and sensitivity, so validation in your specific experimental system is always recommended .

What is the optimal protocol for Western blot detection of DHAR1 protein?

For optimal Western blot detection of DHAR1 protein, follow this methodological approach:

• Extract proteins using an appropriate buffer (e.g., Lyse&Load-Buffer as used in published protocols)

• Separate 10-15 μl of protein extract on a 15% SDS-PAGE gel

• Transfer proteins to PVDF membrane using Bjerrum Buffer in a semi-dry blot system (1 hour transfer time)

• Block membranes with 5% milk in 1× TBS-Tween20 (1%) for 1 hour at room temperature with agitation

• Incubate with primary anti-DHAR1 antibody at 1:5000 dilution (in 5% milk, 1× TBS-Tween20 with 0.01% NaN₃) overnight at 4°C

• Wash three times for 10 minutes each with 1× TBS-Tween20

• Incubate with appropriate secondary antibody (e.g., anti-rabbit IgG AP-conjugate) at 1:2000 dilution for 1 hour

• Wash as above and develop using appropriate detection method (e.g., NBT/BCIP for AP-conjugated antibodies)

This protocol has been validated for detection of DHAR1 in plant samples and provides clear visualization of the expected 23.4 kDa band .

How can researchers validate the specificity of DHAR1 antibodies?

To validate DHAR1 antibody specificity, employ a multi-tiered approach:

• Genetic validation: Use T-DNA insertion lines or knockout mutants (such as dhar1-1, dhar1-2, dhar1-3) as negative controls in Western blots

• Complementation testing: Include complemented lines (e.g., dhar1-3 EOS-DHAR1) to confirm signal restoration

• Cross-reactivity assessment: Test against related proteins (e.g., DHAR2) to evaluate potential cross-reactivity

• Peptide competition assay: Pre-incubate antibody with the immunizing peptide to block specific binding

• Overexpression systems: Use recombinant DHAR1 protein as a positive control

• Multiple detection methods: Confirm findings using different techniques (Western blot, ELISA, immunohistochemistry)

Published studies have used combinations of wild-type samples alongside dhar1 and dhar2 mutants to demonstrate antibody specificity, providing a robust validation approach .

What controls should be included when using DHAR1 antibodies in stress response studies?

When studying stress responses with DHAR1 antibodies, include these essential controls:

• Baseline expression controls: Untreated samples to establish normal DHAR1 expression levels

• Positive induction controls: Samples treated with known DHAR1 inducers (jasmonic acid or oxidative stressors)

• Time-course samples: Multiple time points to capture the dynamics of DHAR1 expression

• Loading controls: Housekeeping proteins (e.g., actin, tubulin) to normalize expression levels

• Genotype controls: Wild-type alongside DHAR1 knockout or knockdown lines

• Related protein controls: Analysis of other DHAR family members to distinguish specific responses

• Pharmacological controls: Antioxidant treatments that may suppress DHAR1 induction

Research has shown that DHAR1 is particularly responsive to salt stress and pathogen infection, as evidenced in studies with tomato plants infected with Verticillium dahliae and mangroves under salt stress .

How can DHAR1 antibodies be employed in understanding subcellular localization and protein interactions?

For advanced subcellular localization and protein interaction studies with DHAR1 antibodies, implement these methodological approaches:

• Immunogold electron microscopy: Use gold-conjugated secondary antibodies against DHAR1 primary antibodies for precise subcellular localization at the ultrastructural level, particularly for confirming mitochondrial localization

• Cellular fractionation: Combine with Western blotting to detect DHAR1 in isolated mitochondria versus cytosolic fractions

• Co-immunoprecipitation (Co-IP): Use DHAR1 antibodies to pull down protein complexes, followed by mass spectrometry to identify interaction partners

• Proximity ligation assay (PLA): Detect protein-protein interactions in situ by combining DHAR1 antibodies with antibodies against suspected interaction partners

• Fluorescence resonance energy transfer (FRET): Use fluorescently labeled DHAR1 antibodies with labeled potential partners to detect close proximity

• Bimolecular fluorescence complementation (BiFC): Complement with genetic approaches to validate interactions detected with antibody-based methods

Understanding DHAR1's subcellular localization is critical as it influences its function in the ascorbate-glutathione cycle and its role in stress response mechanisms .

What considerations should researchers make when developing custom DHAR1 antibodies with specific binding profiles?

When developing custom DHAR1 antibodies with specific binding profiles, consider these advanced parameters:

• Epitope selection: Choose unique peptide sequences that distinguish DHAR1 from other DHAR family members, particularly in the regions that differ from DHAR2

• Cross-specificity design: For antibodies intended to recognize multiple DHAR variants, target conserved regions through computational modeling of binding modes

• Binding mode optimization: Apply biophysics-informed modeling to distinguish between similar epitopes, even when they cannot be experimentally dissociated

• Specificity validation: Employ phage display experiments with systematic variations of CDR3 regions to optimize specificity

• Drug-to-Antibody Ratio (DAR) consideration: For developing therapeutic antibodies or imaging tools, optimize the DAR to balance efficacy and pharmacokinetics

• Post-selection refinement: Use high-throughput sequencing and computational analysis to identify and enhance desired binding characteristics

Recent advances in computational antibody design, including constrained preference optimization methods like those in AbNovo, can help in developing antibodies with both high binding affinity and favorable biophysical properties .

How can researchers interpret contradictory DHAR1 antibody results across different experimental systems?

When facing contradictory DHAR1 antibody results across experimental systems, apply this systematic troubleshooting approach:

• Epitope accessibility analysis: Different fixation or extraction methods may alter epitope exposure; try multiple preparation techniques

• Post-translational modification investigation: DHAR1 may undergo modifications affecting antibody recognition; use phospho-specific or other modification-specific antibodies

• Species-specific sequence divergence: Compare DHAR1 sequences across species to identify potential epitope variations

• Isoform-specific detection: Verify whether the antibody targets specific DHAR1 isoforms that may be differentially expressed

• Context-dependent expression: Stress conditions may alter DHAR1 expression patterns; standardize environmental conditions

• Antibody batch variation: Different lots may have varying specificities; include standardized positive controls

• Statistical analysis: Apply robust statistical methods to determine if differences are significant or within expected variation

For example, contradictory results have been observed in stress response studies, where DHAR1 shows differential regulation depending on the stress type, duration, and tissue examined .

What are the common pitfalls in DHAR1 antibody-based experiments and how can they be mitigated?

Common pitfalls in DHAR1 antibody experiments and their mitigation strategies include:

• Non-specific binding: Pre-adsorb antibodies with plant extracts from knockout lines; optimize blocking conditions using 5% milk or BSA in TBS-Tween20

• Weak signal detection: Increase antibody concentration (e.g., from 1:5000 to 1:2500); extend incubation time; use more sensitive detection systems

• High background: Increase washing duration and frequency; decrease primary and secondary antibody concentrations; ensure fresh blocking agents

• Inconsistent loading: Carefully quantify protein prior to loading; use reliable loading controls; consider stain-free gel technology

• Cross-reactivity with DHAR2: Include dhar2 mutant controls (dhar2-1, dhar2-2) to distinguish specific signals

• Degradation products: Add protease inhibitors to extraction buffers; maintain cold chain throughout sample processing

• Inconsistent transfer: Optimize transfer conditions for the 23.4 kDa molecular weight; consider semi-dry versus wet transfer methods

Studies have shown successful mitigation of these issues through careful optimization of Western blot protocols, particularly in stress response experiments where protein degradation can complicate interpretation .

How should researchers interpret changes in DHAR1 protein levels across different stress conditions?

For accurate interpretation of DHAR1 protein level changes across stress conditions:

• Establish baseline dynamics: Measure DHAR1 levels over time in unstressed conditions to account for natural fluctuations

• Quantify relative changes: Use densitometry with appropriate normalization to housekeeping proteins

• Correlate with enzymatic activity: Complement antibody detection with DHAR1 activity assays to confirm functional significance

• Multi-level analysis: Compare protein levels with transcript abundance to identify post-transcriptional regulation

• Consider redox state: Evaluate whether observed changes reflect altered protein levels or redox-dependent conformational changes

• Pathway integration: Analyze other components of the ascorbate-glutathione cycle simultaneously

• Tissue-specific responses: Different tissues may show opposite responses; clearly define the tissue being analyzed

Research has demonstrated that DHAR1 is particularly responsive in salt stress scenarios, as seen in mangrove studies, and during pathogen infection, as observed in tomato plants challenged with Verticillium dahliae .

What methodological considerations should be made when using DHAR1 antibodies in non-model organisms?

When using DHAR1 antibodies in non-model organisms, apply these methodological considerations:

• Sequence homology assessment: Compare DHAR1 sequences between the non-model organism and the species against which the antibody was raised

• Epitope conservation analysis: Use bioinformatics tools to predict whether the antibody's epitope is conserved

• Gradient titration: Test multiple antibody concentrations to determine optimal dilution for the non-model system

• Cross-reactivity mapping: Test against purified recombinant DHAR proteins from the non-model species

• Preabsorption controls: Use recombinant proteins from the non-model organism to verify antibody specificity

• Sequential epitope exposure: Try multiple antigen retrieval methods to optimize epitope accessibility

• Custom antibody development: Consider developing species-specific antibodies if commercial options show poor reactivity

The demonstrated cross-reactivity of some DHAR1 antibodies between Arabidopsis and tomato suggests potential utility across related plant species, though careful validation is essential .

How can computational models improve DHAR1 antibody design and specificity?

Advanced computational approaches for improving DHAR1 antibody design include:

• Multi-objective antibody design: Apply frameworks like AbNovo that leverage constrained preference optimization to balance binding affinity with other desired properties

• Structure and sequence co-design: Utilize pre-trained antigen-conditioned generative models that simultaneously optimize antibody structure and sequence

• Physical binding energy modeling: Employ continuous rewards rather than pairwise preferences to more accurately model binding interactions

• Primal-and-dual approach: Implement constrained optimization that balances binding affinity as a reward while enforcing explicit constraints on other biophysical properties

• Structure-aware protein language models: Incorporate these models to address limited training data challenges

• Binding mode identification: Use computational models to disentangle different binding modes associated with particular ligands

• Diffusion generative models: Apply these advanced models to generate novel antibody sequences with customized specificity profiles

Recent research demonstrates that such computational approaches can outperform existing methods in metrics of binding affinity (such as Rosetta binding energy), evolutionary plausibility, and biophysical properties like stability and specificity .

What are the latest methodological advances in using DHAR1 antibodies for studying stress response mechanisms?

Recent methodological advances for using DHAR1 antibodies in stress response studies include:

• Single-cell resolution techniques: Combine DHAR1 antibodies with single-cell proteomics to map cellular heterogeneity in stress responses

• In vivo dynamics: Use fluorescently labeled antibody fragments to track DHAR1 localization changes during stress in living cells

• Multi-omics integration: Correlate DHAR1 antibody-based proteomics with transcriptomics and metabolomics for comprehensive pathway analysis

• Temporal proteomic profiling: Apply antibody-based enrichment followed by mass spectrometry to track stress-induced post-translational modifications of DHAR1

• Organelle-specific analysis: Combine subcellular fractionation with DHAR1 immunodetection to track inter-organelle movement during stress

• Protein-metabolite interaction: Use antibody-based pull-down combined with metabolite analysis to understand DHAR1's role in redox metabolite fluctuations

• High-throughput phenotyping: Correlate DHAR1 protein levels with plant phenotypic responses using automated imaging platforms

Research has shown that DHAR1 plays critical roles in the temporal response to vascular wilt pathogens in tomato and in salt stress responses in mangroves, highlighting its importance in diverse stress adaptation mechanisms .

What emerging applications of DHAR1 antibodies are anticipated in plant stress biology?

Emerging applications of DHAR1 antibodies in plant stress biology include:

• Climate adaptation research: Using DHAR1 as a molecular marker for identifying climate-resilient crop varieties

• Biomarker development: Establishing DHAR1 protein levels as diagnostic indicators of specific stress conditions

• CRISPR-based studies: Combining DHAR1 antibodies with gene-edited plant lines to dissect functional domains

• Synthetic biology applications: Engineering modified DHAR1 proteins with enhanced stress protection capabilities

• Field-deployable diagnostics: Developing antibody-based dipstick tests to rapidly assess plant stress status

• Systems biology mapping: Creating comprehensive interaction networks centered on DHAR1 during various stress conditions

• Translational applications: Extending findings from model plants to crops for agricultural improvement

The role of DHAR1 in redox homeostasis under biotic and abiotic stressors positions it as a key target for developing stress-resistant crops, with antibody-based detection methods providing essential tools for both basic research and applied breeding programs .

How can antibody engineering approaches be applied to develop more specific tools for DHAR family protein research?

Advanced antibody engineering approaches for developing more specific DHAR family research tools include:

• Inference-based design: Apply computational models trained on phage display experiments to design antibodies with custom specificity profiles

• Binding mode optimization: Identify distinct binding modes associated with different DHAR family members to enhance specificity

• Cross-specificity engineering: Design antibodies that can selectively bind to conserved epitopes across DHAR family members for comparative studies

• Structure-guided paratope optimization: Use structural information about DHAR proteins to guide antibody paratope design

• Affinity maturation: Apply directed evolution approaches to enhance binding affinity while maintaining specificity

• Fragment-based approaches: Develop smaller antibody fragments with improved tissue penetration for in vivo imaging

• Nanobody development: Create single-domain antibodies with enhanced stability and specificity for DHAR proteins