ERD15 Antibody

What is ERD15 and why is it important in plant research?

ERD15 (Early Responsive to Dehydration 15) is a small, acidic protein that functions as a key stress-responsive factor in plants. It is rapidly induced in response to various abiotic and biotic stress stimuli, including drought, cold, and pathogen infection. ERD15's importance in plant research stems from its role as a negative regulator of abscisic acid (ABA) signaling and its involvement in stress response pathways .
The protein is part of a highly conserved family specific to the plant kingdom, with its origins traceable to the emergence of land plants. In Arabidopsis, ERD15 modulates ABA sensitivity, affecting drought tolerance, freezing tolerance, and disease resistance. In soybean, GmERD15 functions as a novel transcription factor that binds to the NRP-B promoter and activates stress-induced cell death signaling .

What types of ERD15 antibodies are available for plant research?

Based on available research, polyclonal antibodies against ERD15 have been successfully developed and utilized in multiple experimental settings. These antibodies are typically produced by immunizing rabbits with purified ERD15 protein and collecting serum after sufficient immunization .
For research applications, both unmodified anti-ERD15 sera and purified IgG fractions have been employed. While commercial monoclonal antibodies against ERD15 are less commonly reported in the literature, lab-generated polyclonal antibodies have proven effective for applications including western blotting, immunoprecipitation, and chromatin immunoprecipitation (ChIP) assays .

What are the primary applications of ERD15 antibodies in plant science?

ERD15 antibodies serve multiple critical functions in plant science research:

• Protein detection and quantification: Western blot analysis using anti-ERD15 antibodies allows researchers to detect and quantify ERD15 protein levels in plant tissues under various stress conditions .

• Subcellular localization studies: Immunofluorescence and subcellular fractionation coupled with western blotting using anti-ERD15 antibodies have revealed the nucleocytoplasmic distribution of ERD15, suggesting dual functions in different cellular compartments .

• Protein-DNA interaction analysis: In ChIP assays, anti-ERD15 antibodies have been used to demonstrate in vivo binding of ERD15 transcription factors to target promoters such as the NRP-B promoter in soybean .

• Validation of transgenic lines: Anti-ERD15 antibodies are valuable tools for confirming altered ERD15 expression in overexpression or RNAi-silenced plant lines, enabling correlation between protein levels and phenotypic changes .

What is the optimal protocol for generating effective anti-ERD15 polyclonal antibodies?

Based on successful research methodologies, the following protocol is recommended for producing effective anti-ERD15 polyclonal antibodies:
Step 1: Antigen preparation

• Express and purify recombinant ERD15 protein using bacterial expression systems

• Ensure high purity (>90%) through appropriate chromatography techniques

• Verify protein identity through mass spectrometry
  Step 2: Immunization schedule

• Select rabbits as the host animal (most commonly used for anti-ERD15 antibodies)

• Collect pre-immune serum as a control

• Primary immunization: 300 μg of purified ERD15 protein emulsified with complete Freund's adjuvant

• Booster immunizations (3-4): 300 μg of ERD15 with incomplete Freund's adjuvant at 21-day intervals
  Step 3: Antibody collection and validation

• Collect serum one week after the final immunization

• Validate specificity through western blotting using:

  • Recombinant ERD15 protein

  • Plant extracts from wild-type and ERD15-overexpressing lines

  • Pre-immune serum as a negative control

• Test dilution ranges (1:100–1:50,000) to determine optimal working concentration

How should I optimize western blot conditions when working with ERD15 antibodies?

Optimizing western blot conditions for ERD15 detection requires attention to several key parameters:
Sample preparation:

• Extract total protein using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail

• Include 10-20 µg of total protein per lane for plant tissue samples

• For nuclear extracts, use specialized nuclear extraction protocols to ensure proper fractionation
  Electrophoresis conditions:

• Use 12-15% SDS-PAGE gels for optimal resolution of ERD15 (~15-20 kDa)

• Include positive controls (recombinant ERD15) and molecular weight markers
  Transfer and detection:

• Transfer proteins to PVDF or nitrocellulose membranes (100V for 1 hour or 30V overnight)

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature

• Primary antibody: Start with 1:1,000 dilution of anti-ERD15 antibody in blocking buffer, incubate overnight at 4°C

• Secondary antibody: Anti-rabbit IgG conjugated with HRP or alkaline phosphatase at 1:10,000 dilution

• Detection: Use ECL substrate for HRP or NBT/BCIP for alkaline phosphatase
  Recommended controls:

• Plant extracts from ERD15-silenced lines (negative control)

• Pre-immune serum (to identify non-specific binding)

• Loading control (anti-actin or anti-tubulin antibodies)

What methods can be used to study ERD15 subcellular localization with antibodies?

Multiple complementary approaches can be employed to study ERD15 subcellular localization:
Immunofluorescence microscopy:

• Fix plant cells or tissues with 4% paraformaldehyde

• Permeabilize with 0.1% Triton X-100

• Block with 2% BSA in PBS

• Incubate with anti-ERD15 antibody (1:200-1:500 dilution)

• Apply fluorophore-conjugated secondary antibody

• Counterstain nuclei with DAPI

• Visualize using confocal microscopy
  Subcellular fractionation with immunoblotting:

• Isolate cytoplasmic, nuclear, and membrane fractions using differential centrifugation

• Confirm fraction purity using compartment-specific markers (e.g., histone H3 for nuclei)

• Perform western blot analysis using anti-ERD15 antibodies

• Quantify relative distribution across compartments
  Transient expression of tagged ERD15 with antibody validation:

• Generate YFP-ERD15 or similar fusion constructs

• Express in plant cells via Agrobacterium-mediated transformation

• Observe fluorescent protein localization via confocal microscopy

• Validate findings using anti-ERD15 antibodies in parallel immunolocalization studies

• Co-localize with known subcellular markers
  Research has shown that ERD15 exhibits nucleocytoplasmic distribution, being present in both the cytoplasm and nucleus. This dual localization suggests multiple roles for ERD15 in different cellular compartments, with nuclear localization supporting its transcription factor activity despite lacking a conventional nuclear localization signal .

How can ChIP assays with ERD15 antibodies be optimized to study DNA-binding properties?

Chromatin immunoprecipitation (ChIP) with ERD15 antibodies requires careful optimization due to the unique DNA-binding properties of ERD15 and potential technical challenges:
Improved ChIP protocol for ERD15:
Chromatin preparation:

• Cross-link plant tissue with 1% formaldehyde for 10 minutes under vacuum

• Quench with 0.125 M glycine

• Extract nuclei using a hypotonic buffer followed by gradient centrifugation

• Sonicate chromatin to achieve fragments of 200-500 bp (optimize sonication conditions for each tissue type)

• Verify fragmentation efficiency by agarose gel electrophoresis
  Immunoprecipitation:

• Pre-clear chromatin with protein A/G beads and non-immune IgG

• Divide chromatin for immunoprecipitation with:

  • Anti-ERD15 antibody (optimized concentration)

  • Pre-immune serum (negative control)

  • Input sample (positive control)

• Incubate overnight at 4°C with gentle rotation

• Add protein A/G beads and incubate for 2-3 hours

• Perform stringent washing steps (optimize salt concentration)

• Elute protein-DNA complexes and reverse cross-linking
  DNA purification and analysis:

• Treat samples with proteinase K and RNase A

• Purify DNA using phenol-chloroform extraction or commercial kits

• Analyze by qPCR using primers specific to potential binding regions

  • For ERD15, target promoter regions containing the identified 12-bp palindromic binding sequence

  • Include primers for non-target regions as negative controls
    Data analysis:

• Calculate enrichment as percent of input or fold enrichment over control

• Compare binding to different promoter regions

• Correlate binding with gene expression data

What strategies can resolve inconsistent ERD15 antibody results in different plant species?

Researchers working with ERD15 antibodies across different plant species may encounter inconsistent results due to sequence variations, post-translational modifications, or technical factors. The following strategies can help address these challenges:
1. Sequence alignment and epitope analysis:

• Perform sequence alignment of ERD15 proteins from different plant species

• Identify conserved and variable regions

• Design antibodies against highly conserved epitopes for cross-species applications

• Consider using multiple antibodies targeting different epitopes
  2. Species-specific antibody validation:

• Test antibody specificity in each plant species of interest

• Perform western blots with recombinant ERD15 proteins from different species

• Include appropriate controls:

  • ERD15-overexpressing lines

  • ERD15-silenced or knockout lines

  • Pre-immune serum controls
    3. Technical optimization:

• Adjust antibody concentration for each species (typically 1:500-1:2000)

• Modify extraction buffers to account for species-specific differences in protein composition

• Optimize blocking conditions to minimize background

• Consider using enhanced detection systems for low abundance targets
  4. Alternative approaches:

• Use epitope-tagged ERD15 expressed in the species of interest

• Generate species-specific antibodies when cross-reactivity is problematic

• Employ mass spectrometry-based approaches for protein identification and quantification
  Case study: In research comparing Arabidopsis ERD15 and soybean GmERD15, significant functional differences were observed despite some sequence conservation. While the N-terminal PAM2 domain showed high conservation, the C-terminal regions diverged considerably. Arabidopsis ERD15 lacked the putative DNA-binding motif found in GmERD15, explaining why Arabidopsis ERD15 did not bind to the NRP-B promoter in yeast assays. Species-specific antibodies were crucial for accurate characterization of these functional differences .

How can ERD15 antibodies help elucidate protein-protein interactions in stress signaling pathways?

ERD15 antibodies can be powerful tools for investigating protein-protein interactions in stress signaling networks through several methodologies:
Co-immunoprecipitation (Co-IP):

• Prepare plant extracts under non-denaturing conditions to preserve protein interactions

• Immobilize anti-ERD15 antibodies on protein A/G beads or use pre-conjugated antibody beads

• Incubate with plant extracts (consider crosslinking to stabilize transient interactions)

• Wash thoroughly to remove non-specific binding

• Elute bound proteins and analyze by:

  • Western blotting with antibodies against suspected interaction partners

  • Mass spectrometry for unbiased identification of the interactome
    Proximity ligation assay (PLA):

• Fix and permeabilize plant cells or tissue sections

• Incubate with anti-ERD15 antibody and antibody against potential interaction partner

• Apply secondary antibodies conjugated with oligonucleotides

• Perform ligation and rolling circle amplification

• Detect fluorescent signal indicating close proximity (<40 nm) of the proteins
  Bimolecular fluorescence complementation (BiFC) with antibody validation:

• Generate fusion constructs of ERD15 and potential partners with split fluorescent protein fragments

• Express in plant cells via transient transformation

• Observe fluorescence reconstitution by confocal microscopy

• Validate interactions using co-IP with anti-ERD15 antibodies

• Quantify interaction strength through fluorescence intensity measurements
  Research applications:

• Investigation of ERD15 interaction with poly(A)-binding proteins (PABPs) through the PAM2 domain

• Identification of transcriptional complexes formed during stress responses

• Characterization of interactions with components of ABA signaling pathway

• Study of ERD15's role in recruiting transcriptional machinery to target genes
  Research has revealed that ERD15 contains a conserved PAM2 domain that may mediate interactions with poly(A)-binding proteins, suggesting a role in post-transcriptional regulation. Additionally, the nuclear localization of ERD15 despite lacking a conventional nuclear localization signal suggests interaction with nuclear proteins that facilitate its import .

What are common pitfalls when using ERD15 antibodies and how can they be addressed?

Researchers working with ERD15 antibodies may encounter several challenges that can compromise experimental outcomes. Here are common issues and their solutions:
Problem 1: High background in western blots

• Causes: Non-specific binding, insufficient blocking, too high antibody concentration

• Solutions:

  • Increase blocking time (2-3 hours) and use 5% BSA instead of milk for blocking

  • Increase washing stringency (add 0.1% SDS to wash buffer)

  • Optimize antibody dilution (test 1:1000 to 1:5000 range)

  • Pre-absorb antibody with plant extract from ERD15-silenced plants

  • Use more specific secondary antibodies
    Problem 2: Weak or no signal detection

• Causes: Low ERD15 expression, protein degradation, inefficient extraction

• Solutions:

  • Enrich for ERD15 by using stress treatments known to induce expression

  • Include protease inhibitors in all buffers

  • Optimize protein extraction protocol for nuclear proteins

  • Use enhanced chemiluminescence detection systems

  • Consider subcellular fractionation to concentrate ERD15
    Problem 3: Multiple bands in western blots

• Causes: Protein degradation, splice variants, post-translational modifications

• Solutions:

  • Include both N-terminal and C-terminal targeted antibodies to distinguish fragments

  • Use freshly prepared samples and maintain cold chain

  • Add phosphatase inhibitors if phosphorylation is suspected

  • Perform peptide competition assays to identify specific bands
    Problem 4: Poor reproducibility in ChIP assays

• Causes: Insufficient cross-linking, over-sonication, low antibody affinity

• Solutions:

  • Optimize cross-linking time (8-12 minutes)

  • Carefully monitor sonication efficiency

  • Increase antibody amount and incubation time

  • Include positive controls (e.g., known transcription factors)

  • Design primers spanning the 12-bp palindromic sequence identified as ERD15 binding site

How can phosphorylation-specific ERD15 antibodies be developed to study stress-induced post-translational modifications?

Developing phosphorylation-specific antibodies for ERD15 requires careful design and validation to study potentially important regulatory post-translational modifications:
Step 1: Identify potential phosphorylation sites

• Perform in silico analysis using phosphorylation prediction tools (NetPhos, PhosphoSite)

• Analyze mass spectrometry data from phosphoproteome studies

• Consider evolutionary conservation of potential phosphosites across plant species

• Focus on serine, threonine, and tyrosine residues in functional domains
  Step 2: Design phosphopeptide antigens

• Synthesize 10-15 amino acid peptides containing the phosphorylated residue of interest

• Position the phosphorylated residue centrally in the peptide

• Include a terminal cysteine for conjugation to carrier protein

• Synthesize both phosphorylated and non-phosphorylated versions of each peptide
  Step 3: Antibody production

• Conjugate phosphopeptides to KLH or BSA carrier proteins

• Immunize rabbits using the following schedule:

  • Initial immunization with 500 μg conjugated phosphopeptide in complete Freund's adjuvant

  • 3-4 booster immunizations (300 μg each) at 21-day intervals

  • Test bleeds to monitor antibody titer
    Step 4: Purification and validation

• Perform sequential affinity purification:

  • First column: non-phosphorylated peptide to remove antibodies recognizing unmodified protein

  • Second column: phosphorylated peptide to isolate phospho-specific antibodies

• Validate specificity using:

  • ELISA with phosphorylated and non-phosphorylated peptides

  • Western blots with samples treated with/without phosphatase

  • Samples from plants treated with kinase inhibitors

  • Mutated versions of ERD15 (phospho-mimetic and phospho-null mutants)
    Application to ERD15 research:

• Monitor phosphorylation status during different stress conditions

• Correlate phosphorylation with subcellular localization changes

• Investigate how phosphorylation affects DNA-binding capacity

• Study the role of phosphorylation in protein-protein interactions
  While specific information about ERD15 phosphorylation sites is limited in the literature, this approach provides a framework for developing tools to study this important aspect of ERD15 regulation in stress responses.

How can ERD15 antibodies contribute to understanding the evolutionary conservation of stress response mechanisms in different plant species?

ERD15 antibodies can serve as valuable tools for comparative studies exploring the evolutionary conservation of stress response mechanisms across plant lineages:
Cross-species immunological analysis:

• Develop antibodies against highly conserved ERD15 epitopes

• Perform western blot analysis on protein extracts from diverse plant species:

  • Model plants (Arabidopsis, tobacco, rice)

  • Crop species (soybean, maize, wheat)

  • Primitive land plants (mosses, liverworts)

  • Algal species (to establish evolutionary boundaries)

• Correlate immunoreactivity with sequence conservation

• Create a phylogenetic map of ERD15 protein conservation
  Comparative stress response profiling:

• Subject diverse plant species to standardized stress treatments:

  • Drought (PEG treatment or soil water deficit)

  • Cold stress (4°C exposure)

  • Pathogen challenge (using conserved PAMPs)

• Monitor ERD15 protein levels using validated antibodies

• Compare induction kinetics and magnitude across species

• Correlate with physiological stress tolerance parameters
  Functional domain analysis:

• Generate domain-specific antibodies targeting:

  • PAM2 domain (highly conserved)

  • DNA-binding domains (variable between species)

  • C-terminal regions (most divergent)

• Use these antibodies to study functional conservation across species

• Employ immunoprecipitation to identify interacting partners

• Compare these interactomes between species
  Research implications:

• Studies have shown that while the PAM2 domain is highly conserved across plant species, the C-terminal regions show significant divergence

• Functional differences between Arabidopsis ERD15 and soybean GmERD15 have been documented, with GmERD15 possessing transcription factor activity that Arabidopsis ERD15 lacks

• These differences suggest evolutionary divergence in ERD15 function, with conserved roles in stress responses but species-specific mechanisms

• Antibodies that can distinguish these functional variations can help map the evolutionary trajectory of ERD15-mediated stress responses across the plant kingdom

What methodological approaches can resolve contradictory findings in ERD15 function across different plant species?

Contradictory findings regarding ERD15 function across different plant species necessitate integrated approaches to resolve discrepancies:
1. Systematic cross-species functional analysis:

• Combine transcriptomics, proteomics, and metabolomics data from ERD15-modified plants across species

• Use anti-ERD15 antibodies for chromatin immunoprecipitation sequencing (ChIP-seq) to identify binding sites

• Compare binding motifs between Arabidopsis and soybean ERD15

• Correlate binding with gene expression changes

• Apply network analysis to identify conserved and divergent regulatory pathways
  3. Controlled experimental conditions:

• Standardize growth conditions, stress treatments, and developmental stages

• Use identical experimental protocols across species studies

• Employ both genetic (overexpression/silencing) and biochemical (antibody-based) approaches

• Document methodological differences that might explain contradictory results
  4. Resolving specific contradictions:
  The most notable contradiction is between the roles of Arabidopsis ERD15 and soybean GmERD15. While both are stress-responsive, GmERD15 functions as a transcription factor binding to the NRP-B promoter, whereas Arabidopsis ERD15 lacks this DNA-binding capability. This can be resolved by:

• Characterizing the DNA-binding domain present in GmERD15 but absent in Arabidopsis ERD15

• Investigating whether Arabidopsis ERD15 acts as a cofactor in transcriptional complexes despite lacking direct DNA-binding activity

• Exploring alternative mechanisms through which Arabidopsis ERD15 regulates gene expression

How can antibody-based approaches be combined with CRISPR-Cas9 gene editing to advance ERD15 functional studies?

Integrating antibody-based approaches with CRISPR-Cas9 gene editing creates powerful opportunities for precise functional characterization of ERD15:
Strategy 1: Endogenous tagging for improved antibody detection

• Use CRISPR-Cas9 to introduce small epitope tags (FLAG, HA, or Myc) at the N- or C-terminus of the endogenous ERD15 gene

• Validate tag insertion using sequencing and PCR

• Use commercial anti-tag antibodies for enhanced detection sensitivity

• Compare results with native anti-ERD15 antibodies to validate findings

• Benefits: Maintains native expression levels and regulatory elements while enabling sensitive detection
  Strategy 2: Domain-specific functional analysis

• Generate precise domain deletions or modifications using CRISPR-Cas9

• Create plants with modified:

  • PAM2 domains (to disrupt PABP interaction)

  • DNA-binding domains (in species like soybean)

  • Phosphorylation sites

• Use specific anti-ERD15 antibodies to assess protein expression, localization, and interaction patterns

• Compare phenotypes with complete knockout lines
  Strategy 3: Promoter analysis and regulation

• Use CRISPR-Cas9 to modify cis-regulatory elements in the ERD15 promoter

• Employ anti-ERD15 antibodies to quantify resulting protein expression changes

• Correlate with stress sensitivity phenotypes

• Map transcription factor binding sites that regulate ERD15 expression
  Strategy 4: Paralog-specific functional analysis

• In species with multiple ERD15 paralogs, use CRISPR-Cas9 to generate paralog-specific knockouts

• Develop paralog-specific antibodies to monitor compensatory expression changes

• Assess functional redundancy or specialization

• Create comprehensive paralog expression profiles under various stress conditions
  Practical experimental workflow:

• Design and validate CRISPR-Cas9 constructs for ERD15 modification

• Generate and screen edited plant lines

• Confirm modifications at DNA and protein level using specific antibodies

• Challenge plants with stress treatments

• Analyze:

  • Protein expression dynamics using immunoblotting

  • Protein localization using immunofluorescence

  • Protein-protein and protein-DNA interactions using co-IP and ChIP

  • Phenotypic responses to various stresses

• Complement findings with transcriptomic and metabolomic analyses

What future research directions could exploit ERD15 antibodies for crop improvement applications?

ERD15 antibodies can facilitate several promising research directions for enhancing crop stress resilience:
1. High-throughput screening for ERD15 variants associated with stress tolerance:

• Develop antibody-based assays to rapidly screen germplasm collections for ERD15 protein variants

• Correlate ERD15 expression patterns, modification states, and interaction profiles with stress tolerance traits

• Use antibodies to identify naturally occurring ERD15 variants with enhanced or altered function

• Create molecular markers based on identified variants for marker-assisted breeding
  2. Engineering optimal ERD15 expression for balanced stress responses:

• Use antibodies to precisely quantify ERD15 protein levels in engineered crop lines

• Fine-tune ERD15 expression to achieve optimal balance between:

  • Drought/freezing tolerance (requiring ABA sensitivity)

  • Pathogen resistance (potentially compromised by excessive ABA sensitivity)

• Monitor post-translational modifications under different stress combinations

• Develop crops with context-specific ERD15 regulation
  3. Understanding ERD15 function in important crop species:

• Develop species-specific antibodies for major crops (wheat, rice, maize, potato)

• Characterize ERD15 expression, localization, and function across developmental stages

• Investigate crop-specific ERD15 regulatory networks

• Identify unique aspects of ERD15 function in polyploid crop species
  4. Developing diagnostic tools for stress monitoring:

• Create antibody-based biosensors to monitor ERD15 expression/modification in real-time

• Use these tools to:

  • Detect early stress responses before visible symptoms appear

  • Optimize irrigation timing and pathogen management

  • Monitor effectiveness of stress-protective treatments

  • Develop precision agriculture applications
    5. Translating knowledge from model systems to crops:

• Apply knowledge gained from Arabidopsis and soybean studies to crop species

• Use comparative antibody-based studies to identify conserved regulatory mechanisms

• Develop strategies to modify ERD15 function in crops based on mechanistic understanding

• Create crop-specific antibodies targeting key functional domains identified in model systems
  By leveraging ERD15 antibodies in these research directions, scientists can develop crops with enhanced resilience to multiple stresses, addressing critical challenges in agricultural sustainability under changing climate conditions .