ERF071 Antibody

What is ERF071 and why is it significant in plant biology?

ERF071 (also known as HRE2 in some literature) belongs to the ERF-VII subfamily of ERF/AP2 transcription factors. It functions as a key regulator in hypoxic and osmotic stress responses . Experimental evidence shows that ERF-VII proteins like ERF071 bind to specific promoter elements (HRPEs) in hypoxia-responsive genes to control their expression . Understanding ERF071 function is critical for developing crops with enhanced stress tolerance.

What are the best methods for validating ERF071 antibody specificity?

Validation should employ multiple complementary approaches:

• Western blot comparison between wild-type plants and erf071 mutants

• Testing with recombinant protein at multiple concentrations (2.5ng, 10ng, and 25ng)

• Peptide competition assays using the immunizing peptide

• Cross-reactivity assessment with related ERF proteins (particularly ERF71-ERF75)

• Immunoprecipitation followed by mass spectrometry

• ChIP-qPCR targeting known binding sites of ERF071 compared to control regions

What is the recommended western blot protocol for ERF071 antibody?

Based on validated protocols :

• Electrophoresis: 12% SDS-PAGE

• Transfer: Nitrocellulose membrane for 1 hour

• Blocking: 5% skim milk at room temperature or 4°C for 1 hour

• Primary antibody: 1:1000 dilution, overnight at 4°C

• Secondary antibody: 1:10000 dilution using HRP-conjugated anti-rabbit IgG

• Expected molecular weight: 19 kDa

• Detection: Chemiluminescence substrate with CCD camera imaging

How do expression patterns of ERF071 change under stress conditions?

ERF071 expression is highly dynamic during stress responses. After recovery from stress for 3 days, wild-type ERF71-ERF75 expression levels rapidly return to basal levels observed in plants grown under normal conditions . This indicates a tight regulatory mechanism controlling these transcription factors. When designing experiments, consider:

• Time-course sampling (early vs. late stress responses)

• Tissue-specific expression patterns

• Differential expression across various stress types (hypoxia, drought, heat)

• Correlation between transcript and protein levels

How can ERF071 antibodies be used in chromatin immunoprecipitation (ChIP) studies?

For effective ChIP experiments with ERF071 antibodies:

• Optimize crosslinking conditions (1-1.5% formaldehyde for 10-15 minutes)

• Aim for DNA fragmentation of 200-400bp through careful sonication calibration

• Include appropriate controls (IgG, input, and negative genomic regions)

• Design primers for known ERF-VII binding sites as positive controls

• Consider using a dual crosslinking approach if standard protocols yield poor results

• Validate ChIP results with electrophoretic mobility shift assays (EMSA)

Research has demonstrated that related ERF-VII proteins bind to Hypoxia-Responsive Promoter Elements (HRPEs) with the consensus sequence 5′-NCCTGCAT-3′ . When designing ChIP-qPCR primers or analyzing ChIP-seq data, focus on promoter regions containing this motif.

What methodological approach is best for studying ERF071 interactions with other proteins?

Based on studies of related ERFs, consider these approaches:

• Co-immunoprecipitation (Co-IP):

  • Use ERF071 antibodies to pull down protein complexes

  • Analyze by western blot or mass spectrometry

  • Include appropriate controls (pre-immune serum, IgG)

• Yeast two-hybrid validation:

  • Confirm interactions identified by Co-IP

  • Use specific domains of ERF071 to map interaction sites

• BiFC or split-luciferase assays:

  • Visualize interactions in planta

  • Determine subcellular localization of interactions

Research with ERF72 showed interactions with ARF6 and BZR1 using these techniques, revealing its role in hypocotyl elongation . Similar approaches could uncover ERF071's interaction network.

How can ERF071 antibodies be used to study post-translational modifications (PTMs)?

To investigate PTMs of ERF071:

• Immunoprecipitate ERF071 using specific antibodies

• Analyze by mass spectrometry to identify modification sites

• Develop modification-specific antibodies for key PTMs

• Compare PTM patterns across different stress conditions

• Use phosphatase treatments as controls for phosphorylation studies

• Consider 2D gel electrophoresis to separate modified forms

This approach is particularly important as ERF-VII proteins are known to be regulated through the N-end rule pathway, affecting their stability and function .

What approaches can be used to study the relationship between ERF071 and ROS signaling pathways?

Based on findings about related ERF proteins:

• Design experiments that measure:

  • H₂O₂ levels using fluorescent probes

  • Activity of ROS-producing enzymes like RbohD

  • Expression of ROS-responsive genes

• Experimental comparisons:

  • Wild-type vs. erf071 mutants

  • Normal vs. stress conditions

  • Time-course analysis

Research shows that ERF74 (related to ERF071) acts as an "on-off switch" controlling an RbohD-dependent mechanism in response to different stresses, subsequently maintaining hydrogen peroxide homeostasis in Arabidopsis . Similar mechanisms might apply to ERF071.

How can epitope-directed antibody selection improve ERF071 antibody specificity?

Modern antibody development approaches can enhance specificity:

• Epitope-directed selection strategy:

  • Design a directed library favoring target epitopes unique to ERF071

  • Create precise "counter" antigens for clearing irrelevant binders

  • Use structural information to target functionally important regions

• Advantages over conventional methods:

  • Higher specificity for ERF071 vs related ERF proteins

  • Better functional relevance by targeting key domains

  • Reduced cross-reactivity with other plant proteins

This approach has been successfully used for developing highly specific antibodies against targets like FZD2/7 CRD, resulting in antibodies with predictable modulatory activity .

What are common technical challenges when working with ERF071 antibodies and their solutions?

How should samples be prepared for optimal ERF071 detection in different plant tissues?

For optimal results:

• Harvest tissue quickly and flash-freeze in liquid nitrogen

• Use extraction buffer containing:

  • Protease inhibitor cocktail

  • Phosphatase inhibitors (if studying phosphorylation)

  • DTT or β-mercaptoethanol (5-10 mM)

  • PMSF (1 mM)

• Consider nuclear extraction protocols for improved detection

• Normalize protein quantification using Bradford or BCA assays

• Store aliquots at -80°C to avoid freeze-thaw cycles

How can researchers distinguish between ERF071 and other closely related ERF family members?

To ensure specificity:

• Use antibodies raised against unique N-terminal sequences

• Validate with knockout/knockdown mutants for each family member

• Perform parallel RT-qPCR to correlate protein with transcript levels

• Consider using tagged versions of proteins for unambiguous detection

• Pre-absorb antibodies with recombinant proteins of related family members

Research shows that ERF71-ERF75 have overlapping but distinct functions, making specific detection critical for accurate interpretation of results .

What is the optimal experimental design for studying ERF071's role in stress responses?

Based on previous research :

• Genetic materials:

  • Wild-type plants

  • erf071 single mutants

  • erf071/erf075 double mutants (for functional redundancy)

  • ERF071 overexpression lines

• Treatment design:

  • Multiple stress conditions (drought, high light, heat, hypoxia)

  • Time-course sampling (0h, 1h, 3h, 6h, 12h, 24h, 3d recovery)

  • Controlled growth conditions with precise stress parameters

• Key measurements:

  • ERF071 protein levels (western blot)

  • Target gene expression (RT-qPCR, RNA-seq)

  • Physiological parameters (ROS levels, stress tolerance)

  • Chromatin binding (ChIP-qPCR at target promoters)

How can researchers integrate ERF071 antibody data with transcriptomic and genomic approaches?

For comprehensive analysis:

• Multi-omics integration strategy:

  • Correlate ERF071 protein levels with RNA-seq data

  • Compare ChIP-seq profiles with differential expression analysis

  • Use protein-DNA binding data to refine motif analysis

• Validation workflow:

  • Identify potential targets from ChIP-seq/RNA-seq

  • Confirm direct binding by ChIP-qPCR

  • Validate functional significance in knockout/overexpression lines

  • Analyze promoter activity using reporter assays

Research demonstrates that ERF-VII transcription factors bind to evolutionarily conserved promoter elements, information that can guide genomic analyses .

What approaches can reconcile contradictory data between protein levels and phenotypic observations?

When facing contradictory results:

• Technical considerations:

  • Verify antibody specificity in the specific experimental context

  • Examine post-translational modifications affecting protein activity

  • Consider protein localization vs. total protein levels

  • Evaluate functional redundancy with related ERFs

• Biological explanations:

  • Assess threshold effects (minimum protein levels needed for function)

  • Consider time-dependent regulation (early vs. late responses)

  • Evaluate tissue-specific effects that might be masked in whole-plant analyses

  • Examine protein-protein interactions that may modulate activity

How might single-cell approaches enhance ERF071 research using antibodies?

Emerging single-cell technologies offer new research avenues:

• Single-cell western blotting for cell-type-specific ERF071 quantification

• Mass cytometry (CyTOF) with ERF071 antibodies for high-dimensional analysis

• Spatial transcriptomics combined with immunofluorescence for tissue context

• Microfluidic antibody capture for single-cell protein analysis

These approaches could reveal cell-type-specific roles of ERF071 that are masked in whole-tissue analyses.

What new antibody development strategies might improve ERF071 research?

Advanced antibody technologies to consider:

• Single-domain antibodies (nanobodies) for improved intracellular detection

• Recombinant antibody fragments with enhanced specificity

• Antibodies against specific post-translationally modified forms of ERF071

• Multi-specific antibodies for studying ERF071 in protein complexes

• Antibodies optimized for super-resolution microscopy applications

AsEP (Antibody-specific Epitope Prediction) approaches represent a promising direction for developing next-generation antibodies with enhanced specificity for transcription factors like ERF071 .

How can ERF071 antibody research contribute to crop improvement?

Translational applications include:

• Screening germplasm collections for ERF071 protein variants associated with stress tolerance

• Validating gene-edited crops with modified ERF071 expression or activity

• Developing diagnostic tools for monitoring stress responses in crops

• Understanding species-specific differences in ERF071 function between model plants and crops