EDR1 Antibody

What is EDR1 and what are its known functions in different biological systems?

EDR1 has distinct roles in different biological systems:

In mammals, particularly mice, EDR1 (Erythroid Differentiation Regulator 1) is an interleukin 18-regulated gene that functions as a metastasis suppressor in melanoma . The protein is involved in erythroid differentiation processes, though its complete molecular function profile continues to be investigated.

In plants, particularly Arabidopsis thaliana, EDR1 encodes a Raf-like protein kinase similar to CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) . It functions as a negative regulator of several processes:

• Disease resistance signaling pathways

• Salicylic acid (SA)-dependent defense responses

• Abscisic acid (ABA) signaling

• Ethylene-induced senescence

• Stress-induced programmed cell death

Plant EDR1 is involved in the regulation of a MAP kinase cascade (likely including MPK3 and MPK6) and may, together with KEG (KEEP ON GOING), regulate endocytic trafficking and/or the formation of signaling complexes on trans-Golgi network/early endosome vesicles during stress responses .

What types of EDR1 antibodies are currently available for research?

Researchers have access to several types of EDR1 antibodies:

For mammalian research:

• Anti-Mouse Erythroid Differentiation Regulator 1 (Erdr1) monoclonal antibodies, typically derived from rat hosts

• Recombinant EDR1 proteins with His-tags for use as standards or immunogens

For plant research:

• Anti-Serine/threonine-protein kinase EDR1 antibodies that recognize plant EDR1, particularly from Arabidopsis thaliana, Brassica rapa, Brassica napus, and Glycine max

These antibodies are generally available in formats suitable for Western blotting, ELISA, and potentially immunoprecipitation, though applications should be validated for each specific antibody product.

How should EDR1 antibodies be stored and handled for optimal performance?

For optimal performance of EDR1 antibodies, follow these evidence-based protocols:

Storage:

• Short-term (up to 2 weeks): Store at 4°C

• Long-term: Aliquot and store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles as this can lead to protein denaturation and loss of antibody activity

For lyophilized antibodies:

• Use a manual defrost freezer to prevent moisture contamination

• Upon receipt, immediately store at the recommended temperature

Handling during experiments:

• Before use, centrifuge the antibody solution briefly to collect all material at the bottom of the tube

• Keep antibodies on ice during experimental procedures

• Return to appropriate storage conditions promptly after use

• Consider adding sodium azide (0.02%) as a preservative for frequently used aliquots stored at 4°C

These practices will help maintain antibody specificity and activity throughout your research project's duration.

What controls should be included when using EDR1 antibodies in Western blot experiments?

A robust Western blot experiment with EDR1 antibodies requires these essential controls:

Positive controls:

• Recombinant EDR1 protein (if available)

• Lysate from cells/tissues known to express EDR1 (e.g., wild-type Arabidopsis for plant EDR1)

• Overexpression systems (e.g., EDR1-GFP fusion proteins have been successfully used)

Negative controls:

• Lysate from edr1 mutant plants (for plant studies)

• Lysate from tissues not expressing EDR1

• Secondary antibody-only control to detect non-specific binding

Loading and transfer controls:

• Housekeeping protein detection (e.g., actin, tubulin, GAPDH)

• Total protein staining (Ponceau S or similar)

Antibody validation controls:

• Peptide competition assay to confirm specificity

• Multiple antibodies against different epitopes of EDR1 (if available)

Sample preparation considerations:

• For plant EDR1, include protease inhibitors and phosphatase inhibitors as EDR1 functions in kinase signaling pathways

• For membrane-associated proteins like ATL1 that interact with EDR1, appropriate detergent selection is critical

Documented examples show that EDR1-GFP protein has been successfully detected by immunoblotting assays in transgenic plant lines, providing a useful positive control strategy .

How can I validate the specificity of an EDR1 antibody?

Validating EDR1 antibody specificity requires a multi-faceted approach:

• Genetic validation:

  • Compare detection in wild-type vs. edr1 mutant tissues (the gold standard control)

  • Use RNAi knockdown tissues with reduced EDR1 expression

  • Test EDR1 overexpression lines showing increased signal intensity

• Biochemical validation:

  • Peptide competition assay using the immunizing peptide/protein

  • Immunoprecipitation followed by mass spectrometry to confirm the pulled-down protein identity

  • Western blot detection at the expected molecular weight (checking for a single band)

• Orthogonal detection methods:

  • Correlate antibody detection with mRNA expression levels from qPCR or RNA-seq

  • Use multiple antibodies targeting different epitopes within EDR1

  • Compare with tagged versions (e.g., EDR1-GFP) detected via anti-tag antibodies

• Cross-reactivity assessment:

  • Test in tissues from different species to confirm specificity within the expected range of cross-reactivity

  • For plant EDR1 antibodies, test specificity against related MAPKKKs to confirm absence of cross-reactivity

Research has shown that EDR1-GFP genomic constructs under native promoters can complement edr1 mutant phenotypes, making them excellent positive controls for antibody validation while maintaining physiological expression patterns .

What are the optimal conditions for immunoprecipitating EDR1 and its interacting partners?

Optimal immunoprecipitation of EDR1 and its interacting partners requires careful attention to buffer composition and experimental conditions:

Buffer composition:

• For plant EDR1 interactions with MKK4/MKK5: Use buffers containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and standard protease inhibitor cocktail

• For membrane-associated interactions (e.g., EDR1-ATL1): Include 1% digitonin or 0.5-1% NP-40 to solubilize membrane proteins while preserving interactions

• Include phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) to preserve phosphorylation-dependent interactions

Cross-linking considerations:

• For transient interactions, consider a brief formaldehyde cross-linking step (1% for 10 minutes)

• For detecting EDR1-MKK4/MKK5 interactions, cross-linking may be necessary as this interaction has been demonstrated in multiple systems but might be transient in nature

Co-immunoprecipitation strategies:

• For plant studies, co-expression of EDR1 with interacting partners in Nicotiana benthamiana has been successful

• EDR1-GFP fusions have been effectively used for immunoprecipitation using anti-GFP antibodies

• The N-terminal domain of EDR1 is required for interaction with MKK4/MKK5, so antibodies targeting this region may interfere with some protein interactions

Validation methods:

• Reciprocal co-immunoprecipitation (pull down with anti-EDR1 and detect partner, then pull down with partner antibody and detect EDR1)

• Include appropriate negative controls (e.g., non-specific IgG, unrelated protein)

• Confirm interactions using alternative methods like yeast two-hybrid or three-hybrid systems

Research has shown that EDR1 physically interacts with MKK4/MKK5 in plants, and this interaction can be detected by co-immunoprecipitation assays when appropriate conditions are used .

How does EDR1 regulate the MKK4/MKK5-MPK3/MPK6 kinase cascade in plants?

EDR1 employs multiple mechanisms to negatively regulate the MKK4/MKK5-MPK3/MPK6 cascade in plants:

Physical interaction and sequestration:

• EDR1 physically interacts with MKK4 and MKK5 through its N-terminal domain

• This interaction may sequester MKK4/MKK5, preventing their activation or interaction with downstream MPK3/MPK6

• Yeast three-hybrid analyses demonstrated that EDR1 can inhibit the interaction between EDS1 and PAD4, important components in defense signaling pathways

Protein level regulation:

• EDR1 negatively affects MKK4/MKK5 protein levels

• In edr1 mutants, higher levels of MPK3/MPK6 proteins are observed compared to wild-type plants

• This suggests EDR1 may regulate protein stability or expression of these cascade components

Kinase activity regulation:

• edr1 mutants display highly activated MPK3/MPK6 kinase activity

• This activation correlates with enhanced expression of defense-related genes like FRK1, a known target of the MPK3/MPK6 cascade

Genetic evidence:

• Mutations in mpk3, mkk4, or mkk5 suppress edr1-mediated resistance

• Overexpression of MKK4 or MKK5 causes edr1-like resistance and mildew-induced cell death

• These findings establish a clear genetic interaction between EDR1 and the MAPK cascade

The complete mechanism appears to involve both direct protein-protein interactions and regulation of protein levels, collectively fine-tuning plant immune responses to prevent inappropriate or excessive defense activation .

What is the relationship between EDR1 and the salicylic acid signaling pathway?

The relationship between EDR1 and the salicylic acid (SA) signaling pathway is complex and multifaceted:

Genetic dependency:

• Mutations that block or reduce SA production or signaling (such as eds1-1, nim1-1/npr1, pad4-2, and sid2-2) suppress edr1-mediated enhanced disease resistance

• This indicates that edr1-mediated resistance requires an intact SA signaling pathway

SA-dependent gene expression:

• The majority of the 155 genes upregulated during EDS1-PAD4 overexpression are significantly upregulated in edr1 plants compared to wild type after powdery mildew infection

• Gene ontology analysis revealed that these overlapping genes are enriched for processes like SA response and protein phosphorylation

Regulation of EDS1-PAD4 complex:

• EDR1 physically interacts with both EDS1 and PAD4, key components of the SA signaling pathway

• In yeast three-hybrid assays, EDR1 expression inhibits the interaction between EDS1 and PAD4

• Formation of the EDS1-PAD4 heterodimer is essential for transcriptional reprogramming of host defense pathways

Stress response modulation:

• Under drought conditions, edr1 mutants display enhanced stress responses and spontaneous necrotic lesions in an SA-dependent manner

• The drought-induced growth inhibition and spontaneous HR-like phenotypes in edr1 mutants are suppressed by mutations in eds1-1, nim1-1, pad4-2, and sid2-2, but not by ein2-1 (ethylene signaling)

This evidence suggests that EDR1 functions as a negative regulator at the intersection of multiple stress response pathways, with the SA pathway being essential for many edr1-associated phenotypes. EDR1 appears to regulate SA-dependent defense responses partly through modulating the interaction between EDS1 and PAD4, key upstream regulators of SA biosynthesis and signaling .

How can antibody engineering techniques be applied to develop more specific EDR1 antibodies?

Developing highly specific EDR1 antibodies can be achieved through several advanced antibody engineering approaches:

Structure-guided epitope selection:

• Target unique regions of EDR1 by analyzing protein structure data or predictions

• For plant EDR1, focus on regions distinct from other MAPKKKs to avoid cross-reactivity

• Consider epitopes in the N-terminal domain, which is critical for protein-protein interactions but may have unique sequences

Fine-tuning antibody specificity using RFdiffusion:

• RFdiffusion is a recently developed AI platform fine-tuned to design human-like antibodies

• This approach can build antibody loops—the intricate, flexible regions responsible for antibody binding

• It can generate complete antibodies (single chain variable fragments, scFvs) that specifically recognize target epitopes

Rosetta-based antibody design:

• RosettaAntibodyDesign (RAbD) enables both de novo antibody design and affinity maturation

• It allows for sequence optimization based on canonical cluster sequence profiles

• The protocol is highly tunable using CDR instruction files to include/exclude clusters or PDB entries based on requirements

Computational specificity optimization:

• Modern approaches use high-throughput sequencing data and computational analysis to design antibodies with customized specificity profiles

• These methods can identify different binding modes associated with particular epitopes

• Biophysics-informed modeling combined with extensive selection experiments can design antibodies with desired physical properties

Epitope-focused strategies:

• For challenging epitopes, consider FunFolDes (Functional Folding Design), which compensates for motif insertion by adapting the protein backbone

• This approach has been successfully used for structurally irregular epitopes and can be applied to EDR1-specific regions

Validation through multiple assays:

• Test candidate antibodies against wild-type and edr1 mutant samples

• Evaluate cross-reactivity with related proteins (e.g., other MAPKKKs for plant EDR1)

• Perform epitope mapping to confirm binding to the intended region

These advanced techniques can significantly improve the specificity and utility of EDR1 antibodies for research applications across different biological systems.

Why might my EDR1 antibody show inconsistent results in Western blots between experiments?

Inconsistent Western blot results with EDR1 antibodies can stem from several technical and biological factors:

Sample preparation issues:

• Protein degradation: EDR1 may be subject to proteolytic degradation during extraction. Ensure fresh, complete protease inhibitor cocktails are used

• Extraction buffer composition: For plant EDR1, which functions in kinase cascades, include phosphatase inhibitors to preserve phosphorylation states

• Tissue-specific expression: EDR1 expression may vary between tissues and growth conditions, particularly in response to pathogens or stress factors

Antibody-related factors:

• Antibody degradation: Repeated freeze-thaw cycles can diminish antibody activity. Aliquot antibodies upon receipt and store at -20°C or -80°C

• Lot-to-lot variation: Different manufacturing batches may have subtle variations in specificity or potency

• Non-specific binding: Use appropriate blocking conditions (5% non-fat milk or BSA) and validate specificity with edr1 mutant controls

Experimental variables:

• Post-translational modifications: EDR1 is a kinase involved in signaling cascades; its phosphorylation state may affect antibody recognition

• Stress conditions: In plants, EDR1 function is linked to stress responses; sample collection timing after stress application might affect results

• Protein-protein interactions: EDR1 interacts with multiple proteins (MKK4/MKK5, EDS1/PAD4), which might mask epitopes in some extraction conditions

Protocol optimization strategies:

• Standardize tissue collection and processing times

• Try multiple extraction protocols (native vs. denaturing conditions)

• For plant studies, compare samples with and without pathogen treatment or abiotic stress

• Include wild-type and edr1 mutant controls in each experiment

• Consider membrane transfer optimization for high molecular weight proteins

Research has demonstrated that EDR1 protein levels can be affected by experimental conditions and genetic backgrounds, making consistent detection challenging without careful standardization .

How can I distinguish between mammalian and plant EDR1 proteins in cross-system studies?

Distinguishing between mammalian and plant EDR1 proteins in cross-system studies requires careful consideration of their different properties:

Sequence and size differences:

• Mouse EDR1 (ERDR1) has a distinct sequence from plant (Arabidopsis) EDR1, with no significant homology

• Mouse ERDR1 has a calculated molecular weight different from plant EDR1

• Use sequence alignment tools to identify unique regions in each protein that can be targeted by specific antibodies

Antibody selection:

• Use species-specific antibodies: Anti-Mouse ERDR1 antibody for mammalian studies and Anti-Serine/threonine-protein kinase EDR1 antibody for plant studies

• Verify antibody specificity by testing each antibody against both mammalian and plant samples to confirm lack of cross-reactivity

• Consider epitope location: Plant EDR1 antibodies may target the protein kinase domain, which is not present in the mammalian ERDR1

Experimental design:

• Include proper positive controls for each system (mouse tissue lysates vs. Arabidopsis extracts)

• Use recombinant proteins as standards: Mouse ERDR1 (NP_579940.1) and Arabidopsis EDR1 (AT1G08720)

• For Western blots, run mammalian and plant samples side-by-side with molecular weight markers to distinguish based on size

Verification strategies:

• Perform immunoprecipitation followed by mass spectrometry to confirm protein identity

• Use genetic controls (edr1 mutant plants, ERDR1-knockdown mammalian cells)

• Consider tagged versions of each protein (e.g., EDR1-GFP for plants ) as additional controls

Functional distinction:

• Mammalian ERDR1 functions as a metastasis suppressor in melanoma and is regulated by interleukin 18

• Plant EDR1 is a negative regulator of disease resistance and stress responses, functioning in MAP kinase cascades

• Design functional assays specific to each protein's known activities to further distinguish them

These approaches will help researchers accurately distinguish between these functionally distinct proteins that share the same name but have evolved separately in different kingdoms.

What factors might affect EDR1 protein levels in different experimental conditions?

EDR1 protein levels can be influenced by multiple factors that should be considered when designing experiments:

Stress conditions in plants:

• Pathogen infection: Powdery mildew infection can alter EDR1-regulated defense pathways

• Drought stress: EDR1 is involved in drought stress responses, and drought conditions can affect EDR1-dependent phenotypes

• Ethylene exposure: EDR1 negatively regulates ethylene-induced senescence, and ethylene treatment may influence EDR1 function and potentially protein levels

Signaling pathway activation:

• Salicylic acid (SA) signaling: The SA pathway interacts with EDR1 function, and SA levels may influence EDR1 protein regulation

• MAPK cascade activity: EDR1 interacts with and regulates components of the MKK4/MKK5-MPK3/MPK6 cascade, suggesting reciprocal regulation may occur

• Abscisic acid (ABA) signaling: EDR1 is involved in regulating ABA responses, which may affect its stability or expression

Protein turnover mechanisms:

• E3 ubiquitin ligase interaction: EDR1 interacts with E3 ubiquitin ligases like KEEP ON GOING (KEG) and ARABIDOPSIS TOXICOS EN LEVADURA1 (ATL1), suggesting regulation via the ubiquitin-proteasome system

• Protein phosphorylation: As a kinase involved in signaling cascades, EDR1's own phosphorylation state may influence its stability or activity

• Subcellular localization: EDR1 may localize to trans-Golgi network/early endosome vesicles during stress responses, potentially affecting its detection in different cellular fractions

Experimental considerations:

• Tissue selection: Different plant tissues may express varying levels of EDR1

• Developmental stage: EDR1 function in stress responses may vary with plant age

• Growth conditions: Light intensity, photoperiod, temperature, and humidity can all affect stress responses and potentially EDR1 levels

• Extraction method: Membrane association of EDR1 or interacting partners may require specific extraction conditions

Research has shown that overexpression of MKK4 or MKK5 phenocopies edr1 mutation, suggesting complex regulatory relationships between EDR1 and its interaction partners that could affect protein levels in various experimental contexts .

What are the emerging techniques for studying EDR1 protein interactions and modifications?

Emerging techniques for studying EDR1 protein interactions and modifications include:

Advanced proximity labeling approaches:

• BioID and TurboID: Fusing EDR1 with biotin ligase to identify proximal proteins in living cells

• APEX2 proximity labeling: For temporal control of interaction mapping, especially useful for stress-induced interactions

• Split-BioID: For detecting conditional interactions that occur only under specific stress conditions

These methods would be particularly valuable for mapping the dynamic interactions of EDR1 with components of the MAPK cascade and stress response networks in plants .

Cryo-electron microscopy (cryo-EM):

• Single-particle cryo-EM for structural determination of EDR1 complexes with MKK4/MKK5

• Cryo-electron tomography to visualize EDR1 in its cellular context, potentially in association with trans-Golgi network/early endosome vesicles

Advanced mass spectrometry:

• Parallel reaction monitoring (PRM) for targeted quantification of EDR1 phosphorylation sites

• Crosslinking mass spectrometry (XL-MS) to map interaction interfaces between EDR1 and its binding partners

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to analyze conformational changes upon binding to partners or during stress responses

Live-cell imaging techniques:

• FRET-FLIM (Förster Resonance Energy Transfer-Fluorescence Lifetime Imaging Microscopy) for monitoring EDR1 interactions in living cells

• Super-resolution microscopy to visualize EDR1 localization at a nanoscale resolution

• Optogenetic approaches to temporally control EDR1 activity and monitor downstream effects

AI-driven antibody development:

• RFdiffusion for designing highly specific antibodies against different conformational states of EDR1

• Computational approaches to predict and design antibodies with customized specificity profiles for EDR1 epitopes

Single-cell techniques:

• Single-cell proteomics to assess EDR1 levels and modifications in individual cells responding to stress

• Spatial transcriptomics combined with protein detection to correlate EDR1 activity with gene expression patterns in specific cell types

These emerging techniques would significantly advance our understanding of EDR1's dynamic interactions and regulatory mechanisms in both plant and mammalian systems.

How might comparative studies between plant and mammalian EDR1 inform therapeutic developments?

Comparative studies between plant and mammalian EDR1 could yield valuable insights despite their distinct evolutionary origins:

Signaling pathway conservation and divergence:

• While plant EDR1 and mammalian ERDR1 evolved independently, both are involved in stress response regulation

• Plant EDR1 negatively regulates the MAPK cascade , while mammalian ERDR1 suppresses metastasis

• Understanding how these distinct proteins evolved to regulate stress responses could reveal fundamental principles of cellular adaptation

Suppressive mechanisms comparison:

• Plant EDR1 suppresses cell death and defense responses , while mammalian ERDR1 suppresses melanoma metastasis

• Comparing suppressive mechanisms might reveal conserved principles in negative regulation of cellular processes

• This could inform the design of therapies targeting excessive inflammatory responses or cancer metastasis

Therapeutic antibody development:

• Applying advanced antibody engineering techniques developed for plant EDR1 research to mammalian ERDR1

• Designing antibodies that modulate ERDR1 function could potentially enhance its metastasis suppression activity

• RFdiffusion and other AI-driven approaches could accelerate the development of therapeutic antibodies targeting specific EDR1 epitopes

Disease resistance mechanisms:

• Plant EDR1 research has revealed detailed mechanisms of disease resistance modulation

• These insights might inform therapies targeting excessive immune activation in autoimmune diseases

• The EDR1-MKK4/MKK5-MPK3/MPK6 regulatory axis in plants might have parallels in mammalian systems that could be therapeutically relevant

Drug target identification:

• Identifying protein-protein interfaces critical for EDR1 function in plants could inspire the design of small molecule modulators

• These modulators could target analogous interfaces in mammalian systems

• The interaction between EDR1 and E3 ubiquitin ligases in plants suggests potential therapeutic approaches targeting protein stability regulation

By leveraging advanced antibody engineering techniques and applying lessons from plant EDR1 regulatory mechanisms , researchers could develop novel therapeutic approaches for diseases involving dysregulated stress responses or cellular proliferation.

What role might artificial intelligence play in developing next-generation EDR1 antibodies for research applications?

Artificial intelligence is poised to revolutionize EDR1 antibody development through several advanced approaches:

Structure-guided epitope prediction:

• AI algorithms can analyze protein structures to identify optimal epitopes unique to EDR1

• For plant EDR1, this could identify regions that distinguish it from other plant MAPKKKs

• For mammalian ERDR1, epitopes could be selected to avoid cross-reactivity with other secreted proteins

RFdiffusion for antibody design:

• This AI platform specifically designed for antibody development can generate human-like antibodies targeting EDR1

• RFdiffusion specializes in building antibody loops responsible for binding, producing new antibody blueprints unlike any seen during training

• The system can design complete antibodies (scFvs) with optimized binding properties for EDR1 epitopes

Sequence-structure-function relationships:

• AI can analyze large datasets of antibody sequences and their binding properties to identify patterns

• These patterns can inform the design of EDR1 antibodies with enhanced specificity and affinity

• Machine learning models can predict how sequence modifications will affect antibody performance

Specificity profile customization:

• Advanced computational approaches can design antibodies with customized specificity profiles

• Models trained on phage display data can identify different binding modes for similar epitopes

• These techniques can optimize antibodies for either specific binding to particular EDR1 epitopes or cross-specificity for multiple targets

Rosetta-based optimization:

• AI-enhanced versions of tools like RosettaAntibodyDesign can fine-tune antibody properties

• These systems can optimize both sequence and structure for ideal EDR1 binding characteristics

• Integration with experimental data through machine learning improves prediction accuracy

Experimental design optimization:

• AI can analyze experimental conditions and results to identify optimal protocols for antibody characterization

• This reduces the trial-and-error approach traditionally required for antibody development

• Machine learning models can predict which antibody candidates are most likely to succeed in specific applications

Implementation of these AI approaches could significantly accelerate EDR1 antibody development, reduce costs, and improve antibody performance in research applications. The combination of biophysics-informed modeling and experimental validation offers a powerful framework for designing next-generation EDR1 antibodies with precisely tailored properties .