EDR2 Antibody

What is EDR2 and why is it important in plant pathology research?

EDR2 functions as a negative regulator of cell death specifically triggered by pathogen attack and mediated through the salicylic acid defense pathway. The importance of EDR2 lies in its role limiting the initiation of cell death rather than its spread in response to pathogen challenge. EDR2 encodes a novel protein containing a pleckstrin homology domain, a StAR transfer (START) domain, and a plant-specific domain of unknown function (DUF1336) . Research on EDR2 and related antibodies is essential for understanding plant immune regulation, particularly how plants control the fine balance between resistance and destructive cell death responses during pathogen attack .

What are the key structural features of the EDR2 protein that antibodies typically target?

The EDR2 protein contains several distinct domains that serve as potential epitopes for antibody development. These include:

• A pleckstrin homology (PH) domain that binds to phosphatidylinositol-4-phosphate in vitro

• A StAR transfer (START) domain associated with lipid binding/transfer

• A plant-specific domain of unknown function (DUF1336)

When developing or selecting an EDR2 antibody, researchers should consider which domain would be most accessible and immunogenic. The pleckstrin homology domain is often targeted because of its functional significance in membrane targeting, as EDR2:HA:GFP protein localizes to endoplasmic reticulum, plasma membrane and endosomes . Targeting domain-specific epitopes allows researchers to investigate specific functions of different regions of the protein.

How do I properly validate an EDR2 antibody for research applications?

Proper antibody validation is critical for ensuring experimental reproducibility and meaningful results. For EDR2 antibody validation, implement these key steps:

• Specificity testing: Compare wild-type and edr2 mutant plants in Western blots or immunoprecipitation to confirm the antibody detects EDR2 specifically.

• Cross-reactivity assessment: Test the antibody against related proteins (particularly those with similar domains) to ensure specificity.

• Application-specific validation: Validate the antibody separately for each intended application (Western blot, immunofluorescence, ChIP, etc.) .

• Positive and negative controls: Always include proper controls, such as using tissue from edr2 knockout mutants as a negative control .

• Record batch information: Document the manufacturer, catalog number, lot number, and clone ID (for monoclonals) to account for batch-to-batch variability .

Validation should be considered an ongoing process rather than a one-time event, as antibody performance can vary between applications and experimental conditions.

What are the differences between monoclonal and polyclonal EDR2 antibodies in research applications?

The choice between monoclonal and polyclonal EDR2 antibodies significantly impacts experimental outcomes:

Polyclonal antibodies offer advantages when studying EDR2 localization across different cellular compartments (ER, plasma membrane, endosomes) , while monoclonals provide precision when examining specific domains like the pleckstrin homology domain that binds phosphatidylinositol-4-phosphate .

How can I effectively use EDR2 antibodies to investigate protein-protein interactions in the plant immune response?

To effectively study EDR2 protein interactions in plant immunity, consider these methodological approaches:

• Co-immunoprecipitation (Co-IP): Use anti-EDR2 antibodies to pull down EDR2 protein complexes from plant extracts, particularly after pathogen challenge. This can reveal interaction partners that may change during immune activation. Western blot the precipitated complex with antibodies against suspected interacting proteins.

• Proximity labeling: Combine EDR2 antibodies with proximity labeling techniques such as BioID or APEX to identify proteins in close proximity to EDR2 in living cells.

• Bimolecular Fluorescence Complementation (BiFC): While not directly using antibodies, this complementary approach can validate interactions identified through antibody-based methods.

• Protein crosslinking followed by immunoprecipitation: Use chemical crosslinkers to stabilize transient protein interactions before immunoprecipitation with EDR2 antibodies.

• Pull-down assays with subcellular fractionation: Given EDR2's multiple localizations (ER, plasma membrane, endosomes) , perform fractionation before immunoprecipitation to identify compartment-specific interaction partners.

When investigating EDR2 interactions, pay particular attention to components of the salicylic acid signaling pathway, as EDR2's negative regulation of cell death is linked to this pathway . Mutations in genes like pad4, npr1, and eds1 suppress the edr2 phenotype, suggesting functional relationships worth exploring through protein interaction studies .

What approaches can be used to study EDR2 phosphorylation states using antibodies?

Studying EDR2 phosphorylation requires specialized antibody-based techniques:

• Phospho-specific antibodies: Develop or obtain antibodies that specifically recognize phosphorylated residues of EDR2. This may require identifying likely phosphorylation sites through bioinformatics prediction tools or phosphoproteomic analyses.

• Phos-tag SDS-PAGE: Use Phos-tag acrylamide gels followed by Western blotting with EDR2 antibodies to separate phosphorylated from non-phosphorylated forms of EDR2.

• 2D gel electrophoresis: Combine isoelectric focusing with SDS-PAGE followed by immunoblotting with EDR2 antibodies to separate phosphorylated forms.

• Immunoprecipitation followed by phosphoprotein staining: Use EDR2 antibodies for immunoprecipitation, then stain with phosphoprotein-specific dyes or use mass spectrometry to identify phosphorylation sites.

• Proximity ligation assay: Combine EDR2 antibodies with phospho-amino acid antibodies to visualize phosphorylated EDR2 in situ.

Since EDR2 contains a pleckstrin homology domain that binds to phosphatidylinositol-4-phosphate , phosphorylation may regulate this interaction. Thus, studying EDR2 phosphorylation could provide insights into how this protein's membrane association and function are regulated during pathogen response.

How can I design experiments to investigate the relationship between EDR2 and the salicylic acid defense pathway using antibodies?

Designing experiments to study EDR2's relationship with salicylic acid (SA) defense pathways requires careful planning:

• Time-course immunoblotting: Following pathogen challenge, collect samples at multiple time points and perform Western blots with EDR2 antibodies to track protein levels. Compare with SA pathway markers like PR1 .

• Co-localization studies: Use immunofluorescence with EDR2 antibodies alongside antibodies against SA pathway components to examine their spatial relationships during infection.

• Genetic background comparisons: Compare EDR2 protein levels/patterns in wildtype plants versus mutants impaired in SA signaling (pad4, npr1, eds1) using immunoblotting or immunofluorescence.

• Chromatin immunoprecipitation (ChIP): If EDR2 is suspected to interact with transcriptional regulators of the SA pathway, perform ChIP with EDR2 antibodies followed by qPCR for promoters of SA-responsive genes.

• SA treatment experiments: Exogenously apply SA and monitor changes in EDR2 protein levels, post-translational modifications, or localization using appropriate antibody techniques.

Research has shown that PR1 transcript levels (an SA pathway marker) are increased approximately two-fold in edr2-6 compared to wildtype plants after powdery mildew infection . This suggests EDR2 may negatively regulate SA responses, providing a foundation for more detailed mechanistic studies using antibodies.

What are the optimal fixation and antigen retrieval methods for immunolocalization of EDR2 in plant tissues?

Optimizing EDR2 immunolocalization requires careful consideration of fixation and antigen retrieval:

• Fixation options:

  • For preserving membrane structures (where EDR2 localizes): 4% paraformaldehyde combined with 0.1-0.5% glutaraldehyde

  • For better epitope preservation: Ethanol-acetic acid fixation (3:1 v/v)

  • For cryo-sectioning: Flash freezing in OCT compound

• Antigen retrieval methods:

  • Heat-induced epitope retrieval: 10 mM sodium citrate buffer (pH 6.0) at 95°C for 10-20 minutes

  • For membranous structures: Enzymatic treatment with proteinase K (1-5 μg/ml for 5-15 minutes)

  • For heavily cross-linked samples: Combined approach using citrate buffer followed by mild detergent treatment

• Tissue preparation considerations:

  • Section thickness: 5-10 μm for light microscopy, 70-100 nm for immunoelectron microscopy

  • Permeabilization: 0.1-0.5% Triton X-100 or 0.05-0.1% saponin (gentler for membrane proteins)

EDR2 localization to multiple compartments (endoplasmic reticulum, plasma membrane, and endosomes) necessitates careful optimization. Test multiple fixation and retrieval methods, as the optimal approach may vary depending on which EDR2 population you're studying. For example, plasma membrane-localized EDR2 may require different conditions than ER-localized EDR2.

What controls are essential when using EDR2 antibodies in immunoprecipitation experiments?

Rigorous controls for EDR2 immunoprecipitation experiments include:

• Input control: Reserve a portion of the pre-immunoprecipitation lysate to confirm target protein presence.

• Negative controls:

  • IgG control: Use species-matched, non-specific IgG antibodies processed identically

  • Knockout/mutant control: Use edr2 mutant plant tissue as a biological negative control

  • Pre-immune serum control: For polyclonal antibodies, use pre-immune serum from the same animal

• Specificity controls:

  • Peptide competition: Pre-incubate antibody with excess antigenic peptide before immunoprecipitation

  • Isotype control: Use matched isotype antibody targeting an unrelated protein

• Elution controls:

  • Analyze both eluate and post-IP supernatant to assess capture efficiency

  • Include beads-only control to identify non-specific binding to the matrix

• Reverse immunoprecipitation: If studying protein interactions, confirm results by immunoprecipitating the suspected interacting protein and probing for EDR2.

When investigating EDR2's relationship with the salicylic acid pathway, include additional controls such as samples treated with SA pathway inhibitors or activators to distinguish specific from non-specific interactions. Document all antibody information thoroughly, including manufacturer, catalog number, and lot number to enable replication .

How should I optimize Western blot protocols for detecting EDR2 in different plant tissues and subcellular fractions?

Optimizing Western blot protocols for EDR2 detection requires accounting for its multi-compartmental localization and tissue-specific expression:

• Sample preparation optimization:

  • For total protein: Use buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, plus protease inhibitors

  • For membrane fractions: Include 1% NP-40 or 0.1% SDS to solubilize membrane-associated EDR2

  • For intact membrane proteins: Consider using digitonin (0.5-1%) or n-dodecyl-β-D-maltoside (0.5-1%)

• Subcellular fractionation considerations:

  • Separate ER, plasma membrane, and endosomal fractions using sucrose gradient ultracentrifugation

  • Verify fraction purity using markers for each compartment alongside EDR2 detection

• Protein transfer parameters:

  • For complete EDR2 transfer: Use wet transfer at 30V overnight at 4°C

  • Membrane selection: PVDF membranes (0.45 μm) generally work better for EDR2 detection

• Blocking and antibody dilution optimization:

  • Test both BSA and non-fat milk blocking solutions (3-5%)

  • Typical primary antibody dilutions: 1:500 to 1:2000

  • Extended primary antibody incubation: 4°C overnight often yields better results

• Tissue-specific considerations:

  • Leaf tissue: Additional steps to remove phenolic compounds and excess chlorophyll

  • Infected tissue: Compare protein extraction efficiency from healthy versus pathogen-challenged tissue

When working with edr2 mutants as controls, note that these plants develop chlorosis and necrosis at infection sites , which may affect protein extraction efficiency. Consider using uninfected tissue from mutants or sampling at very early infection timepoints to obtain optimal controls.

What are the key considerations for developing a new EDR2 antibody for specific research applications?

Developing a new EDR2 antibody requires strategic planning:

• Epitope selection strategy:

  • Target unique regions to avoid cross-reactivity with related proteins

  • Consider domain-specific antibodies (pleckstrin homology domain, StAR transfer domain, or DUF1336 domain)

  • Avoid regions likely to be obscured by protein-protein interactions or membrane associations

  • Use bioinformatics tools to identify surface-exposed, hydrophilic regions

• Antigen preparation options:

  • Synthetic peptides: 15-20 amino acids long, conjugated to carrier protein

  • Recombinant protein fragments: Express soluble domains for improved immunogenicity

  • Full-length protein: Most challenging but provides most comprehensive antibody response

• Host animal selection factors:

  • Rabbits: Good for polyclonal antibodies with higher yield

  • Mice or rats: Preferred for monoclonal antibody development

  • Consider phylogenetic distance between host animal and plant proteins

• Validation requirements:

  • Western blot against wild-type and edr2 mutant plants

  • Immunofluorescence to confirm expected subcellular localization patterns

  • Pre-absorption tests with immunizing peptide/protein

  • Cross-reactivity testing against related plant proteins

• Application-specific optimization:

  • For co-IP: Focus on epitopes unlikely to be involved in protein-protein interactions

  • For ChIP: Ensure epitope is accessible in crosslinked chromatin

  • For immunofluorescence: Target epitopes that survive fixation procedures

When developing antibodies targeting EDR2's pleckstrin homology domain, consider that this domain binds phosphatidylinositol-4-phosphate , which may affect epitope accessibility in certain experimental conditions.

How can I address contradictory results between different EDR2 antibodies in my research?

Contradictory results between different EDR2 antibodies require systematic troubleshooting:

• Characterize epitope differences:

  • Map the epitopes recognized by each antibody

  • Consider whether different antibodies target different domains of EDR2 (pleckstrin homology, StAR transfer, or DUF1336 domains)

  • Test whether certain epitopes are masked in specific cellular compartments

• Evaluate antibody validation robustness:

  • Re-validate each antibody using knockout/mutant controls

  • Perform peptide competition assays to confirm specificity

  • Check for lot-to-lot variation by testing multiple batches

• Consider post-translational modifications:

  • Determine if contradictory results correlate with different phosphorylation or other modification states

  • Use phosphatase treatment or other enzyme treatments to test this hypothesis

• Reconcile results through complementary approaches:

  • Use epitope-tagged EDR2 constructs to compare with antibody results

  • Apply orthogonal detection methods (mass spectrometry, activity assays)

  • Consider whether different antibodies are detecting different isoforms or degradation products

• Comprehensive reporting:

  • Document all antibody details including catalog numbers and lot numbers

  • Report all contradictory results transparently in publications

  • Include detailed validation methods for each antibody used

When dealing with contradictory results, remember that EDR2 localizes to multiple cellular compartments (ER, plasma membrane, endosomes) and its distribution may change during pathogen response, potentially affecting epitope accessibility.

What are the potential artifacts and pitfalls when using EDR2 antibodies in co-localization studies?

Co-localization studies with EDR2 antibodies present several potential artifacts and pitfalls:

• Fixation-induced artifacts:

  • Over-fixation can cause artificial clustering of membrane proteins

  • Insufficient fixation may allow protein redistribution during processing

  • Different fixatives may preferentially preserve certain EDR2 populations (ER vs. plasma membrane vs. endosomal)

• Antibody cross-reactivity issues:

  • Secondary antibody cross-reactivity in double-labeling experiments

  • Primary antibody non-specific binding to high-abundance plant proteins

  • Cross-reactivity with related proteins containing similar domains

• Resolution limitations:

  • Standard confocal microscopy (resolution ~200-250 nm) may suggest co-localization that's not biologically relevant

  • Super-resolution techniques may be necessary for definitive co-localization at membrane microdomains

• Signal interpretation challenges:

  • Auto-fluorescence from chlorophyll and phenolic compounds in plant tissues

  • Increased background in infected or necrotic tissues (relevant for edr2 mutant studies)

  • Bleed-through between fluorescence channels mimicking co-localization

• Controls and statistical considerations:

  • Need for quantitative co-localization metrics beyond visual assessment

  • Requirement for appropriate spatial randomization controls

  • Importance of biological replicates across different plants/tissues

To minimize these issues, incorporate multiple controls including single-antibody staining controls, secondary-only controls, and wild-type vs. edr2 mutant comparisons . Consider combining co-localization studies with proximity ligation assays or FRET to verify true molecular proximity beyond the resolution limit of conventional microscopy.

How should I interpret changes in EDR2 expression and localization patterns during different stages of pathogen infection?

Interpreting EDR2 dynamics during infection requires careful consideration:

• Timeline correlation analysis:

  • Track EDR2 changes alongside pathogen development stages

  • Compare with established infection phase markers

  • Correlate with the timing of PR1 expression changes (known to increase 2-fold in edr2 mutants)

• Localization pattern analysis framework:

  • Baseline: Document EDR2 distribution across ER, plasma membrane and endosomes in uninfected tissue

  • Early changes: Monitor immediate relocalization upon pathogen perception

  • Late changes: Assess long-term redistribution during sustained infection

• Protein level vs. localization interpretation:

  • Distinguish between changes in total EDR2 protein levels versus redistribution

  • Consider whether changes represent degradation, synthesis, or post-translational modifications

  • Use subcellular fractionation with Western blotting to quantify compartment-specific changes

• Correlation with cell death patterns:

  • Since EDR2 negatively regulates cell death , document the spatial relationship between EDR2 changes and subsequent cell death

  • Develop a temporal map showing EDR2 changes preceding visible cell death symptoms

• Pathogen-specific considerations:

  • Compare responses between different pathogens that trigger EDR2-associated resistance

  • Document differences between compatible and incompatible interactions

  • Note that edr2 mutants show resistance to biotrophic pathogens but not to Pseudomonas syringae

When interpreting EDR2 dynamics, remember that the PR1 transcript (SA pathway marker) shows a two-fold higher induction in edr2-6 compared to wildtype plants after infection , suggesting EDR2 may repress SA-dependent defense responses under normal conditions.

What statistical approaches are most appropriate for quantifying EDR2 antibody staining intensity in immunohistochemistry?

Robust statistical analysis of EDR2 immunohistochemistry requires:

• Appropriate sampling strategies:

  • Random field selection: Minimum 5-10 fields per sample

  • Standardized region selection: Consistent anatomical regions across samples

  • Z-stack sampling: Multiple focal planes to capture 3D distribution

• Quantification methods comparison:

  Method	Advantages	Limitations	Best Application
  Mean fluorescence intensity (MFI)	Simple, widely used	Affected by background	Homogeneous samples
  Integrated density	Accounts for both intensity and area	Sensitive to thresholding	Heterogeneous expression
  Object-based analysis	Counts individual signals	Requires high resolution	Punctate distribution
  Intensity distribution	Captures population heterogeneity	More complex analysis	Mixed cell populations

• Statistical testing approaches:

  • Non-parametric tests (Mann-Whitney U) for intensity comparisons between conditions

  • ANOVA with post-hoc tests for multi-condition comparisons

  • Repeated measures designs for time-course experiments

  • Correlation analysis for co-localization studies

• Control normalization strategies:

  • Internal reference protein normalization

  • Cell area or nuclear staining normalization

  • Background subtraction methods

• Advanced analysis considerations:

  • Machine learning approaches for pattern recognition

  • Bayesian statistics for integrating prior knowledge

  • Spatial statistics for analyzing distribution patterns

What are the most common causes of non-specific binding when using EDR2 antibodies, and how can they be addressed?

Non-specific binding with EDR2 antibodies can arise from several sources:

• Antibody-related factors:

  • Degraded antibody: Store according to manufacturer recommendations; avoid freeze-thaw cycles

  • Excessive concentration: Titrate to determine optimal concentration for specific:non-specific signal ratio

  • Cross-reactivity: Validate against edr2 mutant tissue ; perform preabsorption tests

• Sample preparation issues:

  • Insufficient blocking: Increase blocking agent concentration (5% BSA or milk) and time (2+ hours)

  • Inadequate washing: Implement more stringent wash steps (higher salt, longer duration)

  • Endogenous peroxidase/phosphatase activity: Include appropriate inhibitors in blocking solutions

• Plant tissue-specific challenges:

  • Phenolic compounds binding: Add polyvinylpyrrolidone (PVP) to extraction/blocking buffers

  • Endogenous biotin: For biotin-streptavidin detection systems, include avidin blocking step

  • Wound-induced proteins: Minimize tissue damage during collection and processing

• Protocol optimization strategies:

  • Buffer optimization: Test different detergents (Tween-20, Triton X-100) at various concentrations

  • Temperature adjustments: Compare room temperature vs. 4°C incubation

  • Incubation time balancing: Extend primary antibody incubation while reducing concentration

• Advanced solutions:

  • Antibody purification: Affinity-purify antibodies against recombinant EDR2

  • Cross-adsorption: Pre-incubate with plant extracts from edr2 mutants to remove cross-reactive antibodies

  • Detection system switching: Try alternative systems (e.g., fluorescent vs. enzymatic)

When studying pathogen-infected tissues, remember that edr2 mutants develop chlorosis and necrosis at infection sites , which may increase non-specific binding. Compare infected vs. uninfected tissues carefully to distinguish true signal changes from infection-induced artifacts.

How can I optimize the detection of low-abundance EDR2 protein in different experimental contexts?

Enhancing detection of low-abundance EDR2 requires multilevel optimization:

• Sample enrichment strategies:

  • Subcellular fractionation: Concentrate specific compartments where EDR2 localizes

  • Immunoprecipitation: Concentrate EDR2 prior to analysis

  • Gradient centrifugation: Separate and concentrate membrane fractions

• Protein extraction optimization:

  • Detergent selection: Try CHAPS, digitonin, or octylglucoside for efficient membrane protein extraction

  • Chaotropic agents: Include urea (4-8M) in extraction buffers for comprehensive protein solubilization

  • Protease inhibition: Use freshly prepared, complete protease inhibitor cocktails

• Detection system sensitivity enhancement:

  • Signal amplification: Employ tyramide signal amplification (TSA) for immunohistochemistry

  • Enhanced chemiluminescence: Use high-sensitivity ECL substrates for Western blotting

  • Fluorophore selection: Choose high quantum yield fluorophores for immunofluorescence

• Instrumentation optimization:

  • Exposure time extension: Increase for Western blots while controlling background

  • Detector sensitivity: Use cooled CCD cameras or photomultiplier tubes at optimal settings

  • Microscope settings: Optimize pinhole, gain, and laser power for confocal microscopy

• Negative control importance:

  • Always run parallel detection on edr2 mutant tissue to establish background signal level

  • Process controls identically to ensure valid comparison

For pathogen-responsive studies, consider using pathogen challenges to potentially increase EDR2 expression or alter its distribution pattern. Remember that PR1 expression (a marker of defense activation) increases two-fold in edr2 mutants following infection , suggesting dynamic regulation of defense components that may be useful for timing EDR2 analysis.