DIR15 Antibody

What is DIR15 and why is it important in plant biology research?

DIR15 (Dirigent protein 15) belongs to the dirigent protein family in Arabidopsis thaliana (Mouse-ear cress), which plays significant roles in plant defense mechanisms and lignin biosynthesis. Dirigent proteins are involved in stereoselective coupling of monolignols during lignin formation, contributing to cell wall development and pathogen resistance pathways. Understanding DIR15 function helps elucidate plant stress responses and developmental processes, making it a valuable target for agricultural research aimed at improving crop resilience .

What are the technical specifications of commercially available DIR15 antibodies?

The DIR15 antibody (e.g., CSB-PA203732XA01DOA) is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana DIR15 protein. It is supplied in liquid form containing 50% glycerol and 0.03% Proclin 300 in 0.01M PBS (pH 7.4). The antibody has been affinity-purified and demonstrates specific reactivity with Arabidopsis thaliana samples. It is validated for ELISA and Western blot applications, with recommended dilutions of 1:5000-20000 for ELISA and 1:500-2000 for Western blotting .

How does DIR15 antibody specificity compare to antibodies against other dirigent family proteins?

While specific cross-reactivity data between DIR15 and other dirigent family antibodies is not explicitly provided in the available literature, polyclonal antibodies against plant proteins like DIR15 typically demonstrate some cross-reactivity with closely related family members due to conserved epitopes. Researchers should perform validation experiments using appropriate controls when studying specific dirigent proteins. This might include comparing reactivity patterns in wild-type plants versus knockout mutants or using recombinant proteins of various dirigent family members in competitive binding assays to establish specificity boundaries.

What are the optimal sample preparation protocols for DIR15 antibody in Western blotting?

For optimal Western blot results with DIR15 antibody in Arabidopsis samples, follow this protocol:

• Tissue extraction: Grind 100mg fresh or frozen Arabidopsis tissue in liquid nitrogen to a fine powder.

• Add 500μl extraction buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 1% Triton X-100, 1mM EDTA, protease inhibitor cocktail).

• Centrifuge at 12,000g for 15 minutes at 4°C and collect supernatant.

• Determine protein concentration using Bradford or BCA assay.

• Mix 50μg protein with Laemmli buffer (with β-mercaptoethanol) and heat at 95°C for 5 minutes.

• Separate proteins by SDS-PAGE (10-12% gel recommended).

• Transfer to PVDF or nitrocellulose membrane (25V, 1.3A for 10 minutes using semi-dry transfer).

• Block with 5% non-fat milk in TBST for 1 hour at room temperature.

• Incubate with DIR15 antibody (1:500-1:2000 dilution) overnight at 4°C.

• Wash 3× with TBST, 5 minutes each.

• Incubate with HRP-conjugated secondary antibody (1:10000) for 1 hour at room temperature.

• Wash 3× with TBST, then develop using ECL substrate.

This protocol has been optimized for detecting the ~25-30 kDa DIR15 protein in plant tissue extracts.

How can I design experiments to investigate DIR15 expression during plant stress responses?

To investigate DIR15 expression during plant stress responses, a comprehensive experimental approach should include:

• Stress treatment design:

  • Abiotic stressors: Apply drought (withhold water), heat (38°C), cold (4°C), salt (150mM NaCl), or heavy metals (e.g., 100µM CdCl₂)

  • Biotic stressors: Inoculate with pathogens (Pseudomonas syringae, Botrytis cinerea) or apply pathogen-associated molecular patterns (PAMPs)

  • Control: Maintain plants under standard growth conditions

• Time-course sampling:

  • Collect tissue samples at 0, 3, 6, 12, 24, 48, and 72 hours post-treatment

  • Immediately flash-freeze in liquid nitrogen

• Expression analysis methods:

  • Western blot: Use DIR15 antibody to quantify protein levels

  • qRT-PCR: Design primers specific to DIR15 transcript

  • RNA-seq: For global transcriptome analysis

  • Promoter-reporter constructs: Generate DIR15 promoter:GUS or DIR15 promoter:GFP fusions to visualize tissue-specific expression

• Data analysis:

  • Normalize protein/transcript levels to appropriate housekeeping controls

  • Perform statistical analysis (ANOVA followed by appropriate post-hoc tests)

  • Compare expression patterns across different stressors and timepoints

This experimental design allows for comprehensive assessment of DIR15's role in various stress response pathways.

What immunohistochemistry protocols work effectively with DIR15 antibody for localization studies?

While DIR15 antibody is primarily validated for ELISA and Western blot applications , researchers can adapt the following immunohistochemistry protocol for localization studies:

• Tissue fixation:

  • Fix tissue in 4% paraformaldehyde in PBS overnight at 4°C

  • Rinse with PBS (3 times, 10 minutes each)

  • Dehydrate through ethanol series (30%, 50%, 70%, 95%, 100%, 30 minutes each)

  • Clear with xylene and embed in paraffin

• Sectioning:

  • Cut 5-8μm sections using a microtome

  • Mount on poly-L-lysine coated slides

  • Dry overnight at 37°C

• Immunostaining:

  • Deparaffinize in xylene (3 times, 5 minutes each)

  • Rehydrate through ethanol series (100%, 95%, 70%, 50%, 30%, 5 minutes each)

  • Perform antigen retrieval (10mM sodium citrate buffer, pH 6.0, 95°C, 10 minutes)

  • Block with 5% BSA, 0.3% Triton X-100 in PBS for 1 hour

  • Incubate with DIR15 antibody (1:100-1:200) in blocking solution overnight at 4°C

  • Wash with PBS (3 times, 5 minutes each)

  • Incubate with fluorophore-conjugated secondary antibody (1:500) for 1 hour at room temperature

  • Wash with PBS (3 times, 5 minutes each)

  • Counterstain with DAPI (1μg/ml, 5 minutes)

  • Mount with anti-fade mounting medium

• Controls:

  • Include primary antibody omission controls

  • Use tissue from dir15 knockout mutants as negative controls

  • Consider peptide competition assays to validate specificity

This protocol may require optimization depending on specific tissue types and research questions.

How can I troubleshoot weak or absent signals when using DIR15 antibody in Western blots?

When facing weak or absent signals with DIR15 antibody in Western blots, consider the following troubleshooting strategies:

Additionally, consider protein extraction protocol modifications if DIR15 is membrane-associated or present in specific subcellular compartments. For membrane-associated proteins, include 0.5% sodium deoxycholate in your extraction buffer to improve solubilization.

How do I validate the specificity of DIR15 antibody reactions in my experimental system?

To validate DIR15 antibody specificity, implement these complementary approaches:

• Genetic validation:

  • Compare antibody reactivity in wild-type vs. dir15 knockout mutants

  • Test in DIR15 overexpression lines (expect increased signal)

  • Use CRISPR/Cas9-generated dir15 mutants as negative controls

• Biochemical validation:

  • Perform peptide competition assay: Pre-incubate antibody with excess recombinant DIR15 protein before application (should abolish specific signal)

  • Compare reactivity patterns across tissues/conditions known to have differential DIR15 expression

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

• Analytical validation:

  • Verify molecular weight corresponds to predicted DIR15 size (~25-30 kDa)

  • Assess signal linearity with serial dilutions of sample

  • Compare results with orthogonal techniques (e.g., qRT-PCR for transcript levels)

• Cross-reactivity assessment:

  • Test against recombinant proteins from related dirigent family members

  • Examine antibody performance in species with known DIR15 orthologs

  • Use bioinformatics to identify potential cross-reactive epitopes

Combining these validation strategies provides strong evidence for antibody specificity, enhancing confidence in experimental results.

What are the critical factors affecting DIR15 antibody storage stability and performance?

Several factors significantly impact DIR15 antibody stability and performance:

• Storage conditions:

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

  • Avoid repeated freeze-thaw cycles (more than 3-5 cycles can significantly reduce activity)

  • For frequent use, store a working aliquot at 4°C for up to one month

  • Store in small aliquots (20-50μl) to minimize freeze-thaw events

• Buffer composition:

  • 50% glycerol stabilizes the antibody during freeze-thaw

  • 0.03% Proclin 300 prevents microbial growth

  • pH 7.4 maintains optimal antibody conformation

• Contamination risks:

  • Use sterile technique when handling antibody solutions

  • Avoid introduced proteases that could degrade the antibody

  • Prevent microbial contamination that may produce proteases

• Physical factors:

  • Avoid vortexing (causes protein denaturation through shearing forces)

  • Minimize exposure to light (especially for fluorophore-conjugated antibodies)

  • Maintain cold chain during shipping and handling

• Chemical exposures:

  • Avoid reactive chemicals in the same storage area

  • Protect from oxidizing agents that may damage antibody structure

Optimal storage and handling practices maintain antibody performance throughout its expected shelf life (typically 12 months when properly stored).

How can DIR15 antibody be used to investigate protein-protein interactions in plant defense pathways?

DIR15 antibody can be employed in several sophisticated approaches to study protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Lyse plant tissue in non-denaturing buffer to preserve protein complexes

  • Incubate lysate with DIR15 antibody bound to Protein A/G beads

  • Wash extensively to remove non-specific binders

  • Elute bound protein complexes and analyze by mass spectrometry or Western blot

  • This identifies proteins that directly or indirectly interact with DIR15

• Proximity-based labeling:

  • Generate transgenic plants expressing DIR15 fused to BioID or TurboID

  • These enzymes biotinylate proteins in close proximity to DIR15

  • Use DIR15 antibody to confirm expression/localization of fusion protein

  • Purify biotinylated proteins using streptavidin and identify by mass spectrometry

• Bimolecular Fluorescence Complementation (BiFC):

  • Create fusion constructs of DIR15 and candidate interactors with split fluorescent protein halves

  • Use DIR15 antibody to verify expression levels of fusion proteins via Western blot

  • Visualize interactions through reconstituted fluorescence when proteins interact

• Förster Resonance Energy Transfer (FRET) microscopy:

  • Create fluorescent protein fusions with DIR15 and potential interacting partners

  • Validate expression patterns with DIR15 antibody

  • Measure energy transfer between fluorophores as evidence of close proximity

These approaches can reveal DIR15's role in lignin biosynthesis complexes, stress signaling pathways, and cell wall formation networks.

What strategies can address cross-reactivity concerns when studying closely related dirigent family members?

When investigating specific dirigent family members while managing cross-reactivity concerns, consider these advanced strategies:

• Epitope mapping and antibody engineering:

  • Identify unique epitopes in DIR15 using computational analysis of protein sequences

  • Generate peptide-specific antibodies targeting highly variable regions

  • Perform affinity purification against specific epitopes

  • Test cross-adsorption with recombinant proteins from related family members

• Genetic approaches for validation:

  • Use CRISPR/Cas9 to generate specific dir15 knockout lines

  • Create transgenic plants with epitope-tagged DIR15 (e.g., FLAG, HA, or GFP tags)

  • Generate lines with multiple dirigent gene knockouts to disambiguate signals

  • Employ RNA interference to selectively reduce DIR15 expression

• Advanced analytical techniques:

  • Combine immunoprecipitation with mass spectrometry for definitive protein identification

  • Use quantitative proteomics (e.g., SILAC or TMT labeling) to distinguish true targets

  • Perform 2D gel electrophoresis followed by Western blotting to separate closely related proteins

  • Employ parallel reaction monitoring (PRM) mass spectrometry for specific peptide detection

• Comparative expression analysis:

  • Map distinct expression patterns of dirigent family members across tissues and conditions

  • Use this information to design experiments where DIR15 is uniquely expressed or induced

  • Compare antibody reactivity patterns with known transcript profiles

These approaches collectively enhance specificity when studying individual members of the dirigent protein family.

How can DIR15 antibody be utilized in quantitative proteomics studies of plant stress responses?

DIR15 antibody can be integrated into quantitative proteomics workflows through several sophisticated approaches:

• Immunoaffinity enrichment coupled with mass spectrometry:

  • Immobilize DIR15 antibody on affinity columns or magnetic beads

  • Enrich DIR15 and associated proteins from plant extracts under various stress conditions

  • Process enriched samples for LC-MS/MS analysis

  • Quantify using label-free or labeled methods (TMT, iTRAQ, SILAC)

  • This approach reveals changes in DIR15 abundance and interactome composition during stress

• Selected Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM):

  • Develop specific peptide transitions for DIR15

  • Use DIR15 antibody-based immunoprecipitation to validate MS results

  • Apply targeted proteomics to quantify absolute DIR15 concentrations across conditions

  • Monitor multiple dirigent family members simultaneously with high specificity

• Protein complex analysis:

  • Use DIR15 antibody for affinity purification of protein complexes

  • Apply protein correlation profiling to identify stable and transient interactions

  • Implement crosslinking mass spectrometry to capture direct protein-protein contacts

  • Compare complex composition across stress conditions

• Spatial proteomics integration:

  • Combine immunohistochemistry with laser capture microdissection

  • Apply DIR15 antibody to identify regions of interest

  • Perform region-specific proteomics to map stress response spatial dynamics

  • Correlate with transcriptomics data for multi-omics integration

These methodologies enable researchers to monitor dynamic changes in DIR15 abundance, localization, and interaction networks during plant stress responses with high precision and specificity.

How do I differentiate between specific and non-specific signals when analyzing DIR15 antibody results?

Differentiating specific from non-specific signals requires systematic experimental controls and analytical approaches:

• Essential experimental controls:

  • Negative genetic controls: Compare signals between wild-type and dir15 knockout plants

  • Antibody controls: Include primary antibody omission and isotype control samples

  • Peptide competition: Pre-incubate antibody with excess antigenic peptide to block specific binding

  • Positive controls: Include samples with known DIR15 overexpression

• Signal validation criteria:

  • Molecular weight verification: Confirm signal appears at the expected size (~25-30 kDa for DIR15)

  • Signal pattern consistency: Specific signals should show consistent patterns across replicates

  • Dose-response relationship: Signal intensity should correlate with sample loading amount

  • Physiological relevance: Expression patterns should align with known DIR15 biology

• Advanced analytical approaches:

  • Multiple antibody validation: Test with alternative antibodies targeting different DIR15 epitopes

  • Orthogonal techniques: Confirm with non-antibody-based methods (e.g., mass spectrometry)

  • Signal quantification: Use digital imaging and appropriate software for objective assessment

  • Statistical analysis: Apply appropriate statistical tests to distinguish signal from background

By implementing these strategies, researchers can confidently differentiate specific DIR15 signals from experimental artifacts or cross-reactivity.

What comparative analysis approaches can reveal DIR15 functional insights across different plant stress conditions?

To extract functional insights about DIR15 across diverse stress conditions, implement these comparative analysis frameworks:

• Multi-condition expression profiling:

  • Systematically quantify DIR15 protein levels across different stressors:

    • Abiotic: drought, salt, heat, cold, UV, oxidative stress

    • Biotic: bacterial, fungal, viral pathogens, herbivory

  • Create expression heat maps to visualize condition-specific patterns

  • Cluster responses to identify stress-specific and general stress responses

• Temporal dynamics analysis:

  • Track DIR15 protein levels across detailed time courses

  • Apply mathematical modeling to characterize expression kinetics

  • Compare early vs. late response patterns across stress types

  • Integrate with stress signaling pathway activation timelines

• Correlation network construction:

  • Correlate DIR15 expression with:

    • Other stress-responsive proteins

    • Metabolites (particularly lignin precursors)

    • Physiological parameters (ROS levels, membrane integrity, etc.)

  • Construct protein-protein interaction networks specific to each stress

  • Identify stress-specific shifts in DIR15 interactome composition

• Comparative mutant phenotyping:

  • Assess dir15 knockout phenotypes under various stresses

  • Measure stress tolerance parameters (survival, growth, physiological markers)

  • Compare with wild-type and other dirigent family mutants

  • Quantify lignin content and composition changes using histochemical staining

These approaches collectively reveal DIR15's functional roles across stress conditions, highlighting both conserved and stress-specific functions within plant defense mechanisms.

How should researchers interpret discrepancies between DIR15 protein levels (detected by antibody) and mRNA expression data?

When facing discrepancies between DIR15 protein and mRNA levels, consider these interpretive frameworks:

• Post-transcriptional regulatory mechanisms:

  • miRNA regulation: Investigate whether DIR15 transcripts are targeted by stress-responsive miRNAs

  • mRNA stability: Assess DIR15 transcript half-life under different conditions using transcription inhibition

  • Alternative splicing: Use RT-PCR with primers spanning potential splice junctions to detect isoforms that might not be detected by the antibody

• Translational regulation:

  • Ribosome profiling: Evaluate DIR15 transcript loading onto ribosomes during stress

  • Upstream open reading frames (uORFs): Analyze DIR15 5'UTR for regulatory elements

  • Stress granule sequestration: Investigate whether DIR15 mRNA is sequestered during stress

• Protein stability and turnover:

  • Proteasome inhibition: Test if proteasome inhibitors alter DIR15 levels

  • Protein half-life: Measure DIR15 stability using cycloheximide chase experiments

  • Post-translational modifications: Investigate whether modifications affect antibody recognition

• Technical considerations:

  • Antibody epitope masking: Test whether certain conditions affect antibody accessibility

  • Extraction efficiency: Optimize protein extraction protocols for different conditions

  • Subcellular redistribution: Combine with fractionation to detect localization changes

Understanding these multi-layered regulatory mechanisms helps explain apparent discrepancies and reveals sophisticated stress-responsive control of DIR15 expression beyond transcriptional regulation alone.

What emerging technologies could enhance DIR15 antibody applications in plant molecular biology?

Emerging technologies poised to revolutionize DIR15 antibody applications include:

• Advanced imaging technologies:

  • Super-resolution microscopy: Apply techniques like STORM or PALM with DIR15 antibodies to visualize nanoscale localization and organization

  • Expansion microscopy: Physically expand plant tissue to improve resolution of DIR15 localization

  • Light-sheet microscopy: Enable whole-organ imaging of DIR15 distribution with reduced phototoxicity

  • Correlative light and electron microscopy (CLEM): Combine immunofluorescence with ultrastructural analysis

• Single-cell technologies:

  • Single-cell proteomics: Detect DIR15 in individual cells to reveal cell-type-specific expression

  • Spatial transcriptomics: Correlate DIR15 protein with its transcript in tissue sections

  • Mass cytometry (CyTOF): Multiplex DIR15 detection with dozens of other markers

• Synthetic biology approaches:

  • Nanobody development: Generate plant-optimized nanobodies against DIR15 for in vivo applications

  • Biosensors: Create DIR15 interaction biosensors using FRET or split reporters

  • Optogenetic tools: Develop light-controlled DIR15 variants to manipulate function

• Computational advancements:

  • Machine learning: Apply to image analysis for automated DIR15 pattern recognition

  • AlphaFold-based epitope prediction: Improve antibody design using structural predictions

  • Integrative multi-omics: Develop frameworks to integrate DIR15 antibody data with other -omics datasets

These technologies will enable unprecedented insights into DIR15 dynamics and function in plant defense and development.

How might advanced genetic engineering approaches complement DIR15 antibody studies?

Advanced genetic engineering approaches can synergize with DIR15 antibody studies through:

• Precise genome editing technologies:

  • CRISPR-Cas9 base editing: Create specific amino acid substitutions in DIR15 to study structure-function relationships

  • Prime editing: Introduce precise mutations without double-strand breaks

  • CRISPR interference/activation: Modulate DIR15 expression without permanent genetic changes

  • **Validate antibody specificity using precisely engineered knockouts and modifications

• Sophisticated protein tagging approaches:

  • Split-protein complementation: Engineer DIR15 fusion constructs for in vivo interaction studies

  • Proximity labeling: Fuse DIR15 to TurboID or APEX2 to map its local interactome

  • Degron systems: Create conditionally degradable DIR15 variants for temporal control

  • **Use antibodies to validate expression and localization of engineered constructs

• Multi-gene engineering strategies:

  • Multiplex CRISPR: Simultaneously modify DIR15 and related dirigent family members

  • Synthetic promoter engineering: Create custom expression patterns for DIR15

  • Pathway reconstruction: Transfer entire lignin biosynthesis modules including DIR15

  • **Apply antibodies to monitor expression in reconstructed systems

• Transient expression systems:

  • Agroinfiltration optimizations: Improve transient expression for rapid DIR15 variant testing

  • Cell-free expression systems: Produce DIR15 variants for in vitro studies

  • Protoplast transformation: Enable high-throughput screening of DIR15 variants

  • **Use antibodies for validation and quantification across these systems

These complementary approaches extend beyond traditional antibody applications, enabling mechanistic studies of DIR15 function with unprecedented precision and control.

What computational approaches can enhance DIR15 antibody epitope prediction and cross-reactivity analysis?

Advanced computational methods can significantly improve DIR15 antibody development and application:

• Structural biology integration:

  • AlphaFold2/RoseTTAFold predictions: Generate DIR15 structural models to identify surface-exposed regions

  • Molecular dynamics simulations: Assess epitope flexibility and accessibility

  • Binding site prediction: Identify functional regions that might be conserved across family members

  • Epitope mapping visualization: Create 3D maps of predicted antibody binding sites

• Machine learning approaches:

  • Deep learning epitope predictors: Train on known plant protein epitope data

  • Cross-reactivity prediction: Develop algorithms to predict potential off-target binding

  • Sequence-structure-antibody binding relationships: Build models correlating sequence features with antibody recognition

  • Image analysis automation: Develop tools to quantify immunostaining patterns objectively

• Comparative genomics integration:

  • Dirigent family phylogenetic analysis: Identify unique vs. conserved regions across family members

  • Epitope conservation mapping: Assess conservation of potential epitopes across species

  • Ortholog identification: Predict cross-reactivity with dirigent proteins in other plant species

  • Evolutionary rate analysis: Identify rapidly evolving regions that might serve as specific epitopes

• Integrated database development:

  • Plant antibody epitope database: Collect validated plant protein epitope information

  • Dirigent protein family portal: Centralize sequence, structural, and functional information

  • Cross-reactivity prediction tools: Develop online resources for researchers

  • Wet-lab validation feedback loops: Continuously improve predictions with experimental data

These computational approaches enable more strategic antibody development and application, reducing experimental iterations and improving specificity when studying closely related dirigent family proteins.