ERF017 Antibody

What is ERF017 and what is its biological significance?

ERF017 (Ethylene-responsive transcription factor ERF017) is a member of the DREB subfamily A-5 of the ERF/AP2 transcription factor family. The protein contains one AP2 domain and plays significant roles in plant stress responses. In Arabidopsis thaliana, it is encoded by gene AT1G19210 (also known as T29M8.8 or T29M8_8) . Recent studies have identified a homolog in Cucurbita moschata (pumpkin), CmoERF017, as a positive regulator in response to low-temperature stress, affecting root ABA synthesis and signaling by directly regulating the expression of SDR7 and ABI5 .

ERF017 is particularly important as part of a 15-member subfamily that includes RAP2.1, RAP2.9, and RAP2.10, all implicated in various plant stress responses and developmental processes .

What applications can ERF017 antibodies be used for in plant research?

ERF017 antibodies can be utilized in multiple experimental applications including:

• Western Blotting (WB): For detection and quantification of ERF017 protein expression levels

• Immunohistochemistry (IHC): To visualize the localization of ERF017 in plant tissue sections

• Immunocytochemistry (ICC): For cellular localization studies

• Immunofluorescence (IF): To visualize ERF017 distribution within cells using fluorescent detection

• Flow Cytometry: For quantitative analysis of ERF017 expression at the cellular level

• Immunoprecipitation (IP): For isolating ERF017 and associated protein complexes

• ELISA: For quantitative measurement of ERF017 in plant extracts

The selection of application depends on your specific research question, tissue type, and experimental design.

How should ERF017 antibody samples be stored and handled?

For optimal performance and longevity of ERF017 antibodies:

• Store concentrated antibody solutions at -20°C for long-term storage (up to one year)

• For frequent use and short-term storage (up to one month), store at 4°C

• Avoid repeated freeze-thaw cycles which can compromise antibody functionality

• When handling, maintain cold chain principles whenever possible

• If provided in glycerol solution (common formulation: 1mg/ml in PBS with 0.02% sodium azide, 50% glycerol, pH7.2), ensure proper mixing after thawing

• Always use appropriate safety protocols when handling antibody solutions containing preservatives like sodium azide

These storage guidelines apply to most polyclonal antibodies against ERF017, though specific storage instructions may vary by manufacturer.

What are the recommended protocols for validating ERF017 antibody specificity?

Rigorous validation of ERF017 antibody specificity is essential before experimental use. A comprehensive validation strategy should include:

• Western blot analysis:

  • Use positive control samples (e.g., Jurkat cell lysates have been documented)

  • Include negative controls (tissues/cells known not to express ERF017)

  • Validate band size corresponds to predicted molecular weight (~68 kDa)

  • Consider using recombinant ERF017 protein as additional positive control

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide

  • Compare signal with and without peptide blocking

  • Specific antibodies will show diminished signal when blocked

• Knockout/knockdown validation:

  • Use CRISPR/Cas9 or RNAi approaches to create ERF017-deficient samples

  • Compare antibody reactivity between wild-type and knockout/knockdown samples

• Cross-reactivity assessment:

  • Test against related ERF family members to confirm specificity

  • Especially important since there are 15 members in the DREB subfamily A-5

• Orthogonal method comparison:

  • Compare protein expression with mRNA expression data

  • Utilize RNA-seq or qPCR data to correlate with antibody-based detection

Document all validation steps thoroughly for publication and reproducibility purposes.

How can researchers optimize ERF017 antibody dilutions for different experimental applications?

Optimal antibody dilution varies by application, detection method, and antibody characteristics. For ERF017 antibodies, the following starting dilutions are recommended:

Optimization Protocol:

• Test three dilutions within the recommended range

• Use identical sample preparation methods across dilutions

• Include appropriate positive and negative controls

• Evaluate signal-to-noise ratio, background staining, and specific signal intensity

• For fluorescence applications, assess autofluorescence contribution

• Document optimized conditions for future reproducibility

The optimal dilution is one that provides maximum specific signal with minimal background.

What are the best extraction methods for preserving ERF017 protein integrity in plant samples?

Effective extraction of ERF017 from plant tissues requires careful consideration of buffer composition and extraction conditions:

Recommended Extraction Protocol:

• Buffer Composition:

  • Base buffer: 50mM Tris-HCl pH 7.5, 150mM NaCl

  • Detergent: 0.5-1% Triton X-100 or 0.1-0.5% NP-40

  • Protease inhibitors: Complete protease inhibitor cocktail

  • Phosphatase inhibitors (if studying phosphorylation status)

  • Reducing agent: 1-5mM DTT or β-mercaptoethanol

  • EDTA: 1-2mM to inhibit metalloproteases

• Extraction Procedure:

  • Flash-freeze tissue samples in liquid nitrogen

  • Grind to fine powder using mortar and pestle while maintaining freezing temperature

  • Add extraction buffer at 3-5 ml per gram of tissue

  • Homogenize thoroughly with minimum heat generation

  • Centrifuge at 12,000-15,000g for 15-20 minutes at 4°C

  • Collect supernatant and determine protein concentration

  • Prepare aliquots to avoid freeze-thaw cycles

• Special Considerations for Transcription Factors:

  • Consider nuclear extraction protocols for enrichment

  • Nuclear lysis buffers may improve extraction efficiency

  • DNase treatment may be necessary to reduce viscosity and improve purity

• Storage of Extracts:

  • Store extracted proteins at -80°C for long-term preservation

  • Add glycerol to 10-20% final concentration to prevent freeze damage

  • Analyze samples as soon as possible after extraction

This protocol maximizes the recovery of active ERF017 protein while minimizing degradation.

How can researchers determine if ERF017 antibodies cross-react with homologs from other plant species?

Cross-reactivity assessment is critical for studies comparing ERF017 across species or when working with non-model plants. A systematic approach includes:

• Sequence homology analysis:

  • Perform multiple sequence alignment of ERF017 protein sequences from target species

  • Identify conservation in the epitope region recognized by the antibody

  • Calculate percent identity and similarity in key regions

• Western blot validation:

  • Test identical protein amounts from multiple species

  • Include positive control (Arabidopsis thaliana samples)

  • Document molecular weight variations across species

  • Analyze band patterns for specificity

• Epitope competition assay:

  • Design peptides representing potential cross-reactive regions

  • Test antibody binding with and without peptide competition

  • Quantify relative binding affinity across species

• Recombinant protein testing:

  • Express ERF017 homologs from different species

  • Compare antibody reactivity in controlled conditions

  • Calculate relative detection sensitivity

• Known homolog testing:

  • The antibody reactivity data between Arabidopsis ERF017 and pumpkin CmoERF017 can provide insight into cross-reactivity patterns

  • Document cross-reactivity with related transcription factors within the DREB subfamily

A thorough comparison table documenting cross-reactivity can be valuable for publication and collaborative research efforts.

How can ERF017 antibodies be used to study transcription factor dynamics during plant stress responses?

ERF017 antibodies can provide valuable insights into transcription factor behavior during stress responses through multiple experimental approaches:

• Chromatin Immunoprecipitation (ChIP) Assays:

  • Use ERF017 antibodies to precipitate DNA-protein complexes

  • Identify direct binding targets through ChIP-seq or ChIP-qPCR

  • Map genome-wide binding profiles under different stress conditions

  • Compare binding sites during normal vs. stress conditions (e.g., cold stress)

• Co-Immunoprecipitation (Co-IP) Studies:

  • Identify protein interaction partners during stress responses

  • Compare interaction networks between normal and stress conditions

  • Validate specific interactions with known stress response factors

• Time-Course Expression Analysis:

  • Monitor ERF017 protein levels at different time points following stress exposure

  • Correlate protein expression with transcriptional changes of target genes

  • Assess post-translational modifications under stress conditions

• Nuclear-Cytoplasmic Fractionation:

  • Track subcellular localization changes during stress response

  • Quantify nuclear accumulation kinetics following stress

  • Correlate localization with transcriptional activity

• Pulse-Chase Experiments:

  • Determine protein turnover rates during stress adaptation

  • Compare stability under normal vs. stress conditions

• Immunofluorescence Microscopy:

  • Visualize spatial distribution changes during stress

  • Co-localize with other stress-responsive factors

  • Track relocalization in response to signaling events

This multi-faceted approach allows comprehensive characterization of ERF017's role in stress response pathways, particularly in cold tolerance mechanisms as identified in recent CmoERF017 studies.

What approaches can be used to investigate the relationship between ERF017 and hormone signaling pathways?

Investigating ERF017's role in hormone signaling requires integrative experimental approaches:

• Hormone Treatment Studies:

  • Treat plant tissues with hormones (ethylene, ABA) at various concentrations

  • Monitor ERF017 protein levels via Western blot with anti-ERF017 antibodies

  • Assess changes in subcellular localization using immunofluorescence

  • Compare wild-type response to ERF017 overexpression/knockout lines

• ChIP-seq After Hormone Treatment:

  • Perform chromatin immunoprecipitation with ERF017 antibodies following hormone exposure

  • Identify differential binding sites after hormone treatment

  • Map binding motifs to hormone-responsive elements

  • Correlate with transcriptomic changes in hormone response genes

• Protein-Protein Interaction Networks:

  • Use ERF017 antibodies for co-IP followed by mass spectrometry

  • Identify interactions with known hormone signaling components

  • Validate key interactions through bimolecular fluorescence complementation

  • Construct interaction networks under different hormone treatments

• Transcriptional Reporter Assays:

  • Use ERF017 antibodies to confirm binding to promoters of interest

  • Correlate binding with reporter gene expression

  • Test effects of hormone pathway mutants on ERF017-dependent transcription

  • Assess cooperative effects with other transcription factors

• Targeted Analysis of ERF017-Hormone Connections:

  • Focus on ABA signaling components based on CmoERF017 research

  • Validate direct regulation of ABI5 and SDR7 in different plant systems

  • Test effects of ABA biosynthesis inhibitors on ERF017 activity

This systematic approach will provide mechanistic insights into how ERF017 integrates into hormone signaling networks, particularly in stress response pathways.

What strategies can be employed for developing ERF017 antibodies with improved specificity for challenging applications?

Developing highly specific ERF017 antibodies requires strategic approaches:

• Epitope Selection Strategies:

  • Analyze sequence alignment of all 15 DREB subfamily A-5 members

  • Identify regions unique to ERF017 outside the conserved AP2 domain

  • Prioritize sequences with high antigenicity and surface exposure

  • Consider multiple epitopes for comprehensive coverage

• Advanced Antibody Engineering Approaches:

  • Generate recombinant antibody fragments (Fab, scFv) for improved tissue penetration

  • Apply phage display selection with negative selection steps against related family members

  • Implement affinity maturation to enhance binding specificity

  • Consider intrabody development for subcellular targeting

• Validation with Advanced Controls:

  • Create CRISPR/Cas9 knockout lines of ERF017

  • Generate cell lines with controlled expression of ERF017 variants

  • Develop epitope-tagged ERF017 reference materials

  • Test against panels of related transcription factors

• Application-Specific Antibody Development:

  • For ChIP applications: optimize for native epitope recognition

  • For microscopy: select epitopes accessible in fixed samples

  • For in vivo tracking: develop non-interfering binding sites

  • For proximity labeling: create fusion-compatible antibody formats

• Cross-Species Considerations:

  • Generate pan-species antibodies targeting highly conserved regions

  • Develop species-specific antibodies for comparative studies

  • Validate across evolutionarily diverse plant families

  • Document cross-reactivity profiles comprehensively

These approaches can significantly improve antibody performance in challenging applications such as ChIP-seq, live-cell imaging, and studies of low-abundance transcription factor variants.

How can ERF017 antibodies contribute to understanding transcription factor evolutionary conservation and divergence?

ERF017 antibodies offer powerful tools for evolutionary studies of transcription factor functions:

• Cross-Species Immunoprecipitation:

  • Test ERF017 antibody reactivity across diverse plant species

  • Perform IP-MS to identify interaction partners in different species

  • Compare complex composition to infer functional conservation/divergence

  • Document differences in post-translational modifications

• Chromatin Binding Conservation Analysis:

  • Conduct ChIP-seq in multiple species using validated cross-reactive antibodies

  • Compare binding motifs and genomic targets across evolutionary distance

  • Identify core conserved targets versus species-specific targets

  • Correlate binding patterns with phenotypic adaptations

• Function-Structure Relationship Studies:

  • Immunoprecipitate ERF017 from different species

  • Analyze protein modifications and structural features

  • Correlate with functional differences in transcriptional activity

  • Map conservation of regulatory domains

• Developmental Expression Profiling:

  • Use antibodies for immunohistochemistry across diverse species

  • Compare tissue/cell-specific expression patterns

  • Document developmental timing differences

  • Correlate with evolutionary adaptations

• Phylogenetic Applications:

  • Generate comparative data on ERF017 epitope conservation

  • Create antibody reactivity profiles across phylogenetic distance

  • Correlate with sequence-based phylogenies

  • Identify potential neofunctionalization or subfunctionalization events

This approach provides functional evidence to complement sequence-based evolutionary analyses of plant transcription factor families.

What methods can be used to investigate ERF017 involvement in cold stress tolerance pathways?

Based on recent discoveries about CmoERF017's role in cold tolerance, several approaches can be employed:

• Temperature-Dependent Binding Studies:

  • Use ERF017 antibodies for ChIP-seq at different temperatures

  • Identify temperature-sensitive binding targets

  • Map cold-responsive element (CRE) motifs bound by ERF017

  • Compare with other cold-responsive transcription factors

• Protein Complex Dynamics:

  • Immunoprecipitate ERF017 under normal and cold conditions

  • Identify temperature-dependent interaction partners

  • Analyze complex formation kinetics during temperature transitions

  • Test stability of interactions under varying temperature conditions

• Genetic Complementation with Antibody Validation:

  • Create ERF017 knockout/knockdown lines

  • Complement with wild-type or mutant versions

  • Use antibodies to confirm expression levels

  • Correlate with cold tolerance phenotypes

  • Validate direct regulation of SDR7 and ABI5 as identified in CmoERF017 studies

• Hormone Signaling Integration:

  • Investigate ERF017 protein levels after ABA treatment

  • Compare with cold-induced expression changes

  • Use antibodies to track nuclear localization during cold stress

  • Correlate with activation of ABA-responsive genes

• Tissue-Specific Expression Analysis:

  • Use immunohistochemistry to map ERF017 expression in different tissues during cold stress

  • Focus on root tissues where ABA signaling is critical

  • Compare with ethylene response pathways

  • Document cell-type specific responses

These approaches can validate and extend the findings from CmoERF017 studies to other plant species and provide mechanistic insights into cold tolerance pathways.

How can researchers use ERF017 antibodies in combination with transcriptomic approaches for integrated analysis?

Integration of antibody-based protein studies with transcriptomics offers powerful insights:

• ChIP-seq and RNA-seq Integration:

  • Perform ChIP-seq using ERF017 antibodies to identify binding sites

  • Conduct parallel RNA-seq to determine expression changes

  • Integrate datasets to identify direct transcriptional targets

  • Create comprehensive regulatory networks

• Proteomics-Transcriptomics Correlation:

  • Use antibodies to quantify ERF017 protein levels

  • Compare with mRNA expression data from RNA-seq

  • Identify post-transcriptional regulatory mechanisms

  • Document protein-mRNA correlations during stress responses

• Single-Cell Applications:

  • Combine single-cell transcriptomics with antibody-based imaging

  • Map cell-type specific expression patterns

  • Correlate with single-cell RNA-seq data

  • Identify heterogeneous responses within tissues

• Time-Course Integration:

  • Track ERF017 protein expression kinetics with antibodies

  • Perform parallel RNA-seq at matching timepoints

  • Document delays between transcriptional activation and protein accumulation

  • Model temporal regulatory networks

• Validation of QTL Studies:

  • Use antibodies to confirm expression of candidate genes identified in QTL studies

  • Correlate protein expression with phenotypic variation

  • Validate functional candidates from RNA-seq studies

  • Example from CmoERF017 research where RNA-seq and QTL-seq were integrated to identify a key cold tolerance gene

What are common challenges in ERF017 antibody-based experiments and how can they be addressed?

Researchers frequently encounter several challenges when working with ERF017 antibodies:

• Low Signal Intensity:

  • Cause: Insufficient antibody concentration, low target abundance, or protein degradation

  • Solution: Optimize antibody concentration, enrich nuclear fraction for transcription factors, include protease inhibitors, and use fresh samples

• High Background Signal:

  • Cause: Non-specific binding, insufficient blocking, or cross-reactivity

  • Solution: Increase blocking time/concentration, optimize antibody dilution, perform additional washing steps, pre-adsorb antibody with non-specific proteins

• Inconsistent Results:

  • Cause: Variable sample preparation, antibody degradation, or technical inconsistencies

  • Solution: Standardize protocols, aliquot antibodies to avoid freeze-thaw cycles, include internal controls in every experiment

• Poor Reproducibility in ChIP Experiments:

  • Cause: Inefficient chromatin shearing, suboptimal crosslinking, or epitope masking

  • Solution: Optimize sonication conditions, test different crosslinking times, verify antibody compatibility with fixed samples

• Cross-Reactivity with Related Proteins:

  • Cause: Conserved domains across the ERF family

  • Solution: Validate specificity with knockout controls, use peptide competition assays, consider raising antibodies against unique regions

• Quantification Challenges:

  • Cause: Variable loading, inconsistent transfer, or detection limitations

  • Solution: Use loading controls, implement internal standards, consider quantitative methods like ELISA or automated Western blot systems

• Low Immunoprecipitation Efficiency:

  • Cause: Weak antibody affinity, improper conditions, or low target abundance

  • Solution: Increase antibody amount, optimize buffer conditions, extend incubation time, use more starting material

Documentation of troubleshooting steps is essential for methods sections and laboratory records.

How can researchers effectively evaluate batch-to-batch variation in ERF017 antibodies?

Ensuring consistency between antibody batches is critical for experimental reproducibility:

• Standard QC Panel Testing:

  • Develop a standard panel of positive and negative control samples

  • Test each new batch against this panel using your primary application

  • Document signal intensity, background levels, and specific/non-specific bands

  • Calculate batch-to-batch variation coefficient

• Quantitative Comparison Protocol:

• Epitope Binding Validation:

  • Perform competitive binding assays with immunizing peptide

  • Compare inhibition curves between batches

  • Test cross-reactivity profiles with related proteins

  • Document any shifts in epitope preference

• Application-Specific Testing:

  • Validate each batch specifically for your intended application

  • For ChIP applications, compare peak profiles and enrichment values

  • For imaging, evaluate background and specific signal intensity

  • For proximity labeling, compare labeling efficiency

• Documentation and Record-Keeping:

  • Maintain detailed records of batch numbers and performance metrics

  • Create antibody validation files with standardized testing results

  • Document any adaptation required for new batches

  • Consider including batch validation data in publications