ERF096 Antibody

What is ERF96 and why is it important in plant research?

ERF96 is a small ethylene response factor transcription factor belonging to the Group IX ERF family in Arabidopsis thaliana. It contains 131-139 amino acids and is characterized by the presence of an AP2/ERF domain and a CMIX-1 motif of unknown function . ERF96 is significant in plant research because it functions as a transcriptional activator involved in multiple stress response pathways:

• It positively regulates abscisic acid (ABA) responses in Arabidopsis, affecting seed germination, seedling development, and root elongation .

• ERF96 enhances plant resistance to necrotrophic pathogens like Botrytis cinerea and Pectobacterium carotovorum .

• It activates the expression of jasmonate (JA) and ethylene (ET)-responsive defense genes by directly binding to GCC elements in their promoters .

Understanding ERF96 function provides insights into plant hormone signaling and stress response mechanisms, making antibodies against this protein valuable research tools.

How is ERF96 structurally and functionally related to other ERF proteins?

ERF96 belongs to a subfamily of small ERFs in Arabidopsis that includes ERF95, ERF96, ERF97, and ERF98. These four ERFs share 68-88% similarity and 47-74% identity at the amino acid level . Phylogenetic analysis shows that ERF95, ERF96, and ERF97 form a close cluster, while ERF98 is more distantly related .

All four small ERFs contain the AP2/ERF DNA binding domain, but they have distinct expression patterns and functions:

• ERF95 (also called ESE1) and ERF98 are involved in salt tolerance regulation .

• ERF97 (previously named AtERF14) and ERF96 both regulate plant defense responses .

• Uniquely, ERF96 contains an EDLL motif at its C-terminus (amino acids 105-131) that is responsible for its transcriptional activation activity .

This structural and functional diversity highlights the importance of using highly specific antibodies when studying individual ERF proteins to avoid cross-reactivity.

What tissue-specific expression patterns does ERF96 exhibit?

ERF96 shows a distinctive tissue-specific expression pattern in Arabidopsis. Quantitative RT-PCR analysis revealed that:

• Relatively high expression of ERF96 occurs in seeds and flowers .

• Moderate expression is present in stems, leaves, and siliques .

• ERF96 transcript is undetectable in roots .

This expression pattern differs from the other three small ERF genes (ERF95, ERF97, and ERF98), which show different tissue distributions . When designing experiments using ERF96 antibodies, researchers should consider these expression patterns to determine appropriate tissue samples for analysis.

How do ERF96 antibodies help elucidate transcriptional regulatory mechanisms?

ERF96 antibodies are instrumental in deciphering the transcriptional regulatory mechanisms through several advanced techniques:

• Chromatin Immunoprecipitation (ChIP) assays allow researchers to identify direct binding targets of ERF96 in the genome. This has revealed that ERF96 directly binds to GCC elements in the promoters of defense genes including PDF1.2a, PR-3, PR-4, and the transcription factor ORA59 .

• Co-immunoprecipitation (Co-IP) experiments using ERF96 antibodies help identify protein-protein interactions between ERF96 and other transcription factors or components of the transcriptional machinery, revealing regulatory complexes.

• Immunolocalization studies confirm that ERF96 primarily localizes to the nucleus, consistent with its function as a transcription factor .

These techniques have contributed to understanding how ERF96 functions as an activator of transcription in the JA/ET signaling pathways, enhancing plant resistance to necrotrophic pathogens.

What considerations should be made when interpreting ERF96 antibody data in mutant or overexpression studies?

When working with ERF96 antibodies in genetic manipulation studies, several factors should be carefully considered:

• In ERF96 overexpression studies, protein abundance may not linearly correlate with phenotypic effects. For example, overexpression of ERF96 results in hypersensitivity to ABA, smaller rosette size, and delayed flowering, but the magnitude of these effects depends on complex regulatory networks .

• In ERF96-RNAi knockdown plants, basal expression levels of defense genes like PDF1.2 are reduced, yet these plants may still demonstrate wild-type resistance to necrotrophic pathogens due to functional redundancy with other ERF transcription factors .

• When analyzing ERF96 protein levels in hormone treatment experiments, consider that ERF96 expression is regulated by both JA and ET. ERF96 transcript accumulation is abolished in JA-insensitive coi1-16 and ET-insensitive ein2-1 mutants , which may affect antibody-detected protein levels.

• Cross-reactivity with other small ERFs (ERF95, ERF97, and ERF98) should be carefully controlled for, given their high sequence similarity.

How can contradictions in ERF96 function be investigated using antibody-based approaches?

Researchers sometimes encounter contradictory data regarding ERF96 function. Antibody-based approaches can help resolve these contradictions:

• Dual immunodetection can determine if ERF96 forms different protein complexes under various stress conditions, explaining context-dependent functions.

• Comparative ChIP-seq analysis using ERF96 antibodies under different stress conditions (drought, pathogen infection, hormone treatments) can reveal condition-specific binding patterns.

• Phospho-specific ERF96 antibodies can detect post-translational modifications that might explain functional differences in various contexts. This is particularly relevant as transcription factor activity is often regulated by phosphorylation.

• Sequential ChIP (re-ChIP) can determine if ERF96 co-occupies promoters with different partner proteins depending on the stimulus, explaining how the same protein mediates different responses.

These approaches have helped reconcile observations that ERF96 functions in both ABA signaling and pathogen defense pathways by revealing distinct regulatory mechanisms under different conditions.

What are the optimal fixation and extraction protocols for ERF96 immunodetection?

For effective ERF96 immunodetection in plant tissues, the following optimized protocols should be considered:

• Protein Extraction: Best results are achieved using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM DTT, and protease inhibitor cocktail. This composition preserves ERF96 structure while minimizing interference from plant compounds.

• Tissue Fixation for Immunohistochemistry: For optimal ERF96 epitope preservation, use 4% paraformaldehyde in PBS for 2 hours at room temperature, followed by gradual dehydration and paraffin embedding.

• Nuclear Protein Extraction: Since ERF96 is primarily nuclear-localized, using a nuclear extraction protocol significantly improves detection sensitivity. This involves tissue homogenization in nuclear isolation buffer, filtering through miracloth, and nuclear protein extraction with high-salt buffer.

• Sample Preparation for ChIP: Crosslink plant material with 1% formaldehyde for exactly 10 minutes at room temperature, as over-fixation can mask the ERF96 epitope recognized by antibodies.

These protocols minimize the common challenges of background signals and non-specific binding that can complicate plant transcription factor immunodetection.

What validation strategies confirm ERF96 antibody specificity?

Validating ERF96 antibody specificity is critical given the high sequence similarity with other small ERFs. Multiple complementary approaches are recommended:

• Western blot analysis using recombinant ERF95, ERF96, ERF97, and ERF98 proteins to assess cross-reactivity. An ideal ERF96 antibody should show minimal recognition of the other ERF proteins despite their sequence similarities.

• Immunoprecipitation followed by mass spectrometry confirms the identity of the pulled-down protein as authentic ERF96.

• Immunostaining comparison between wild-type plants and erf96 knockout mutants verifies that the observed signal is truly ERF96-specific.

• Antibody pre-absorption tests with recombinant ERF96 protein demonstrate that pre-incubation eliminates specific staining in immunohistochemistry applications.

• Epitope mapping using a series of truncated ERF96 constructs identifies which regions of the protein are recognized by the antibody, helping predict potential cross-reactivity.

These validation steps are especially important for distinguishing between the four small ERF proteins that share 68-88% similarity at the amino acid level .

How can ERF96 antibodies be optimized for chromatin immunoprecipitation (ChIP) studies?

Optimizing ERF96 antibodies for ChIP studies requires specific considerations:

• Epitope Selection: Antibodies raised against the C-terminal EDLL domain (amino acids 105-131) may be superior for ChIP as this region is exposed when ERF96 binds DNA through its AP2/ERF domain.

• Crosslinking Conditions: Optimizing formaldehyde concentration (0.75-1%) and crosslinking time (8-12 minutes) is crucial for maintaining ERF96-DNA interactions without overfixing.

• Sonication Parameters: Using 10-15 cycles of 30 seconds on/30 seconds off at medium power typically yields optimal chromatin fragments of 200-500 bp for ERF96 ChIP.

• Antibody Concentration: Titrating antibody amounts from 2-10 μg per ChIP reaction helps identify the optimal concentration that maximizes signal-to-noise ratio.

• Sequential ChIP Protocol: For studying ERF96 interactions with other transcription factors like ORA59, a sequential ChIP approach can be employed using antibodies against both proteins.

These optimizations have enabled researchers to confirm direct binding of ERF96 to GCC elements in the promoters of defense genes including PDF1.2a, PR-3, and PR-4 .

How can ERF96 antibodies be used to study hormone crosstalk in stress responses?

ERF96 antibodies provide powerful tools for investigating hormone crosstalk in plant stress responses:

• Dual Immunofluorescence: Simultaneous detection of ERF96 and other transcription factors (like ABI5 for ABA signaling or ORA59 for JA/ET signaling) reveals potential co-localization and cooperative action during stress responses.

• ChIP-seq Analysis following different hormone treatments (ABA, JA, ET) identifies hormone-specific and overlapping ERF96 binding sites, providing a genome-wide view of ERF96-mediated transcriptional reprogramming.

• Protein Complex Analysis: Immunoprecipitation of ERF96 followed by mass spectrometry under different hormone treatments identifies hormone-specific ERF96 interaction partners.

• Phosphorylation State Monitoring: Using phospho-specific ERF96 antibodies to track post-translational modifications in response to different hormones helps elucidate how ERF96 activity is regulated by multiple signaling pathways.

These approaches have revealed that ERF96 functions at the intersection of ABA and JA/ET signaling pathways, enhancing both abiotic stress tolerance and resistance to necrotrophic pathogens .

What techniques combine ERF96 antibodies with transcriptome analysis for functional studies?

Integrating ERF96 antibodies with transcriptome analysis creates powerful approaches for functional characterization:

• ChIP-seq followed by RNA-seq: This approach identifies direct ERF96 binding targets and correlates them with differentially expressed genes, distinguishing primary from secondary effects of ERF96 activity.

• RNA Immunoprecipitation (RIP): Using ERF96 antibodies for RIP can identify any RNA molecules that might associate with ERF96, potentially revealing additional regulatory mechanisms.

• Translating Ribosome Affinity Purification (TRAP): When combined with ERF96 promoter-driven expression, this technique allows tissue-specific assessment of translational changes regulated by ERF96.

• Single-cell Approaches: Combining ERF96 immunodetection with single-cell RNA-seq can reveal cell-type-specific functions of ERF96 in complex tissues.

Application of these integrated approaches has shown that ERF96 enhances the expression of defense genes such as PDF1.2a, PR-3, PR-4, and the transcription factor ORA59 by direct binding to GCC elements in their promoters .

How do modifications to the ERF96 protein affect antibody recognition and experimental interpretation?

Post-translational modifications and protein interactions can significantly affect ERF96 antibody recognition and experimental outcomes:

• Phosphorylation Effects: ERF96 likely undergoes phosphorylation during stress responses, which may mask or expose epitopes. Researchers should consider using phosphatase treatments in parallel samples to address this variable.

• Protein-Protein Interactions: When ERF96 forms complexes with other proteins, antibody epitopes may become inaccessible. Using mild detergents or alternative fixation methods can help preserve antibody recognition in co-IP experiments.

• Conformational Changes: The binding of ERF96 to DNA through its AP2/ERF domain may induce conformational changes that affect antibody recognition. This is particularly relevant for ChIP applications where epitope availability in the DNA-bound state is crucial.

• Degradation Products: During stress responses, ERF96 may undergo partial degradation. Antibodies recognizing different regions of the protein can help distinguish between full-length and truncated forms.

• Cross-reactivity Under Different Conditions: The high sequence similarity between ERF96 and other small ERFs (ERF95, ERF97, and ERF98) means that experimental conditions that denature proteins may increase cross-reactivity. Native conditions often preserve structural differences that enhance specificity.

Understanding these factors is essential for accurate interpretation of experimental results using ERF96 antibodies in different contexts.