ERF106 Antibody

What is ERF106 and what is its functional role in plants?

ERF106 functions as a transcriptional activator that specifically binds to the GCC-box pathogenesis-related promoter element. It plays a critical role in regulating gene expression in response to various stress factors and is an important component of stress signal transduction pathways in plants, particularly Arabidopsis thaliana. ERF106 belongs to the ethylene response factor (ERF) family, which is part of the AP2/ERF superfamily of transcription factors.

When studying ERF106 function, researchers should employ multiple complementary approaches:

• Gene expression analysis under various stress conditions

• Chromatin immunoprecipitation (ChIP) to identify DNA binding sites

• Protein-protein interaction studies to identify regulatory partners

• Transgenic approaches (overexpression or knockout) to evaluate phenotypic effects

ERF106 is encoded by the gene AT5G07580 in Arabidopsis, with corresponding database identifiers including KEGG: ath:AT5G07580, STRING: 3702.AT5G07580.1, and UniGene: At.26419. These identifiers are valuable for database searches and comparative analyses with other ERF family members.

How should researchers validate the specificity of ERF106 antibodies?

Validating antibody specificity is crucial for ensuring reliable experimental outcomes when studying ERF106. Based on approaches used with related ERF proteins, a comprehensive validation strategy should include:

• Genetic validation: Test antibody reactivity in erf106 knockout or knockdown plant lines, which should show absent or significantly reduced signal compared to wild-type plants.

• Recombinant protein testing: Express and purify recombinant ERF106 with affinity tags (His-tag or FLAG-tag) and validate detection using both the ERF106-specific antibody and tag-specific antibodies. Sequential purification using His-tag columns followed by anti-FLAG antibody affinity gel can yield highly purified protein for validation purposes .

• Cross-reactivity assessment: Test the antibody against closely related ERF family members, particularly ERF6 and ERF96, which share structural and functional similarities . This is essential given the high sequence similarity within the ERF family.

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide before application in detection assays. Specific signals should be eliminated or substantially reduced.

• Western blot analysis: Evaluate antibody specificity by protein size, expecting a single band of appropriate molecular weight in wild-type samples and no band in knockout samples.

What experimental approaches are recommended for studying ERF106 phosphorylation?

Based on studies of related ERF proteins, particularly ERF6, phosphorylation likely represents a key regulatory mechanism for ERF106 activity. To effectively study ERF106 phosphorylation:

• In vitro kinase assays: Purify recombinant ERF106 and test phosphorylation with candidate kinases, particularly MPK3 and MPK6, which have been shown to phosphorylate the related ERF6 protein . Use radioactive ATP (γ-32P-ATP) or phospho-specific detection systems to visualize phosphorylation.

• Phosphorylation site prediction and mapping: Analyze the ERF106 sequence for potential MAPK phosphorylation sites (Ser/Thr-Pro motifs). Generate Ser/Thr to Ala mutants for each candidate site and combinations thereof, following the approach used for ERF6, where researchers designated putative phosphorylation sites with numbers and systematically mutated them to identify crucial residues .

• Mobility shift detection: Monitor changes in protein migration on SDS-PAGE following treatments that induce phosphorylation. Phosphorylated proteins typically show reduced electrophoretic mobility compared to non-phosphorylated forms. Include phosphatase treatment controls to confirm that observed shifts are phosphorylation-dependent .

• In vivo phosphorylation studies: Generate transgenic plants expressing tagged versions of ERF106 (e.g., myc-tagged) under either native or constitutive promoters. Subject these plants to different stress conditions and analyze protein extracts for phosphorylation status, as was successfully done with ERF6 following Botrytis cinerea infection .

How do ERF106 antibodies compare to antibodies against other ERF family members?

When comparing antibodies directed against different ERF family members, researchers should consider both structural similarities and functional distinctions:

• Epitope selection considerations: ERF proteins share highly conserved DNA-binding domains but differ in other regions. Antibodies raised against the conserved AP2/ERF domain may cross-react with multiple family members, while those targeting unique N- or C-terminal regions typically offer greater specificity.

• Cross-reactivity profiles: ERF106 antibodies should be extensively tested against closely related ERFs, particularly ERF6, which has been shown to be phosphorylated by MPK3/MPK6 , and ERF96, which regulates plant resistance to necrotrophic pathogens .

• Application-specific performance: An antibody that performs well in Western blotting may not necessarily work for immunoprecipitation or ChIP applications. Each application requires specific validation.

• Epitope accessibility considerations: Post-translational modifications or protein-protein interactions may mask antibody epitopes in certain experimental contexts. For instance, phosphorylation of ERF6 causes a detectable mobility shift , which might affect epitope recognition.

• Tag-based detection alternatives: For challenging applications, epitope-tagged versions of ERF106 can be expressed and detected using well-characterized commercial antibodies against tags like myc, FLAG, or HA, similar to the approach used with 4myc-tagged ERF6 .

What are the optimal conditions for using ERF106 antibodies in chromatin immunoprecipitation (ChIP) experiments?

Optimizing ChIP protocols with ERF106 antibodies requires careful consideration of several factors, based on experience with related ERF proteins like ERF96 :

• Crosslinking optimization: Test multiple formaldehyde concentrations (1-3%) and incubation times (5-20 minutes) to effectively capture ERF106-DNA interactions without over-crosslinking, which can reduce antibody accessibility or increase background.

• Sonication parameters: Carefully optimize sonication conditions to generate DNA fragments of 200-500 bp. Inadequate sonication results in low resolution, while excessive sonication can destroy epitopes recognized by the antibody.

• Antibody selection and validation: Validate the ERF106 antibody specifically for ChIP applications, as antibodies that perform well in Western blot may be unsuitable for ChIP. Consider polyclonal antibodies for their ability to recognize multiple epitopes, increasing the chance of detection in crosslinked chromatin.

• Controls and normalization:

  • Include positive controls targeting promoters with known GCC-box elements, similar to those used in ERF96 studies

  • Use promoter regions lacking GCC-boxes as negative controls

  • Include ChIP with pre-immune serum or IgG as procedural controls

  • Normalize to input DNA and preferably also to a consistently expressed reference gene

• Targeted vs. genome-wide approaches: For initial characterization, ChIP-qPCR targeting predicted binding sites in candidate genes is recommended. For comprehensive binding site identification, ChIP-seq offers genome-wide coverage but requires careful bioinformatic analysis to identify enriched motifs.

• Binding site verification: Confirm direct binding to identified loci using electrophoretic mobility shift assays (EMSA) or reporter gene assays with wild-type and mutated GCC-box elements.

How can researchers differentiate between specific and non-specific binding when using ERF106 antibodies?

Distinguishing specific from non-specific binding is critical for accurate interpretation of ERF106 antibody data. Methods to address this challenge include:

• Sequential purification strategies: Implement multi-step purification processes similar to those used for ERF6, where His-tag column purification was followed by anti-FLAG antibody affinity gel purification to achieve high specificity . This approach significantly reduces non-specific contamination.

• Competitive binding assays: Pre-incubate antibodies with excess immunizing peptide or recombinant ERF106 protein before application in the experimental system. Specific signals should be substantially reduced or eliminated.

• Multiple antibody validation: When available, use antibodies raised against different epitopes of ERF106. Consistent results with multiple antibodies provide stronger evidence for specific binding.

• Genetic controls: Compare results between wild-type plants and erf106 knockout/knockdown lines. Signals present in both genotypes likely represent non-specific binding.

• Stringency optimization: Systematically test increasing salt concentrations and detergent levels in wash buffers to determine conditions that maximize the specific-to-non-specific signal ratio.

• Cross-linking optimization: For ChIP experiments, optimize formaldehyde cross-linking conditions to preserve specific interactions while minimizing random cross-links that contribute to background.

• Quantitative analysis: Apply statistical approaches to distinguish significant signals from background noise, establishing clear thresholds for positive identification.

What methodological approaches can resolve contradictory data when analyzing ERF106 binding specificity?

When facing contradictory results regarding ERF106 binding specificity, researchers should implement a systematic troubleshooting approach:

• Antibody characterization and standardization:

  • Verify epitope recognition through peptide array analysis or epitope mapping

  • Compare antibody lots through standardized validation protocols

  • Consider purifying antibodies using antigen-affinity chromatography

  • Establish internal reference standards for quantitative comparisons

• Experimental design optimization:

  • Implement blinded experimental designs to minimize bias

  • Increase biological and technical replicates

  • Standardize sample preparation protocols across experiments

  • Apply appropriate statistical tests to evaluate significance of observed differences

• Orthogonal validation approaches:

  • Use alternative detection methods such as mass spectrometry

  • Compare results with CRISPR-engineered epitope-tagged endogenous ERF106

  • Apply antibody-independent approaches (e.g., DNA affinity purification followed by mass spectrometry)

  • Compare binding profiles with closely related ERFs like ERF6 and ERF96

• Context-dependent binding investigation:

  • Evaluate binding under different physiological conditions

  • Assess the impact of post-translational modifications on binding specificity

  • Investigate potential co-factors that might influence binding patterns

  • Examine cell-type or tissue-specific differences in binding profiles

• Systematic bias identification:

  • Test multiple antibody concentrations and incubation conditions

  • Evaluate the influence of different blocking agents and buffer compositions

  • Assess the impact of sample processing methods on epitope availability

The methodological approach used for ERF6 phosphorylation analysis, where different mutant constructs were systematically tested, provides a useful framework for resolving contradictions in binding specificity data .

How can researchers optimize immunoprecipitation protocols for ERF106 to identify novel interaction partners?

Optimizing immunoprecipitation (IP) protocols for ERF106 requires careful consideration of multiple factors:

• Sample preparation optimization:

  • Test different tissue types and developmental stages

  • Evaluate various stress conditions that might induce relevant interactions

  • Optimize cell lysis and nuclear extraction conditions to maintain protein complexes

  • Consider reversible crosslinking to stabilize transient interactions

• Antibody selection and validation:

  • Use affinity-purified antibodies specific to ERF106

  • Consider epitope tagging approaches (myc, FLAG, HA) similar to those used for ERF6

  • Validate antibody performance specifically for IP applications

• IP conditions optimization:

  • Test different binding buffers varying in salt concentration, detergents, and pH

  • Optimize antibody concentration and binding incubation times

  • Compare various wash conditions to remove non-specific interactions while preserving specific ones

  • Evaluate different elution methods (competitive elution with peptides, pH elution, boiling in SDS)

• Controls implementation:

  • Include IgG control immunoprecipitations

  • Perform IP from erf106 knockout/knockdown material

  • Create tagged versions of ERF106 with mutations in key functional domains

• Detection and identification strategies:

  • Use highly sensitive mass spectrometry for comprehensive partner identification

  • Apply quantitative approaches (SILAC, TMT labeling) to distinguish specific from non-specific interactions

  • Implement bioinformatic filtering based on known subcellular localization and function

• Validation of identified interactions:

  • Confirm key interactions through reciprocal co-IP

  • Verify functional relevance using yeast two-hybrid or split-luciferase assays

  • Assess co-localization through fluorescence microscopy

  • Evaluate phenotypic effects of disrupting specific interactions

The sequential purification approach applied to ERF6, combining His-tag column purification and anti-FLAG antibody affinity gel, provides an excellent model for achieving high specificity in ERF106 interaction studies .

What strategies can detect subtle conformational changes in ERF106 resulting from phosphorylation?

Detecting subtle conformational changes in ERF106 resulting from phosphorylation requires sophisticated biophysical and biochemical techniques:

• Mobility shift analysis:

  • Perform high-resolution SDS-PAGE to detect subtle migration differences

  • Use Phos-tag acrylamide gels to enhance separation of phosphorylated species

  • Compare migration patterns before and after phosphatase treatment

  • Apply two-dimensional gel electrophoresis to resolve complex phosphorylation patterns

• Limited proteolysis:

  • Compare digestion patterns of phosphorylated and non-phosphorylated ERF106

  • Identify regions with altered accessibility to proteases

  • Map cleavage sites using mass spectrometry to identify conformationally altered regions

• Spectroscopic methods:

  • Apply circular dichroism (CD) to detect changes in secondary structure

  • Use intrinsic fluorescence to monitor changes in the local environment of tryptophan residues

  • Implement nuclear magnetic resonance (NMR) for residue-specific conformational analysis

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Compare hydrogen-deuterium exchange rates between phosphorylated and non-phosphorylated forms

  • Identify regions with altered solvent accessibility or hydrogen bonding

  • Map conformational changes to specific functional domains

• Structural analysis:

  • When possible, determine high-resolution structures using X-ray crystallography

  • Apply cryo-electron microscopy for larger complexes

  • Use small-angle X-ray scattering (SAXS) to detect global conformational changes

  • Implement computational modeling to predict conformational effects of phosphorylation

• Functional assays:

  • Compare DNA binding affinity before and after phosphorylation

  • Assess protein-protein interaction profiles with and without phosphorylation

  • Evaluate transcriptional activation capacity as a function of phosphorylation status

These approaches, particularly when used in combination, can provide comprehensive insights into how phosphorylation alters ERF106 conformation and function, similar to the effects observed with ERF6 phosphorylation by MPK3/MPK6 .

Comparative Analysis of ERF Family Members