ERF061 Antibody

What is ERF061 and why are antibodies against it important for research?

ERF061 is a member of the Ethylene Response Factor (ERF) family of transcription factors that play pivotal roles in plant immune responses. Specifically in maize, ZmERF061 has been identified as a member of the B3 group in the ERF family and functions as a nucleus-localized transcription activator that specifically binds to the GCC-box element . ERF proteins contain a highly conserved DNA-binding domain (DBD or AP2 domain), which consists of approximately 57-59 amino acid residues .

Antibodies against ERF061 are crucial for:

• Detecting protein expression levels during pathogen response

• Investigating subcellular localization patterns

• Studying post-translational modifications that regulate activity

• Examining protein-protein interactions, particularly with MAPK pathway components

• Characterizing the protein's role in various stress responses

Research has shown that ZmERF061 expression is significantly induced by pathogen inoculation (particularly Exserohilum turcicum) and hormone treatments with salicylic acid (SA) and methyl jasmonate (MeJA), indicating its importance in plant defense pathways .

How does ERF061 function in plant immunity and what role do antibodies play in elucidating this function?

ERF061 functions as a transcription factor that regulates defense-related genes in response to pathogen attack. In maize, ZmERF061 knockout mutant lines demonstrated enhanced susceptibility to Exserohilum turcicum by decreasing the expression of defense genes ZmPR10.1 and ZmPR10.2 and reducing the activity of the antioxidant defense system .

Antibodies play critical roles in understanding ERF061 function by:

• Tracking protein accumulation during infection progression

• Identifying regulatory mechanisms such as phosphorylation

• Confirming binding to promoter regions of target genes

• Verifying interactions with signaling components like MPK6

Similar to other ERF proteins, ERF061 likely functions downstream of MAPK cascades, as demonstrated by the interaction between ZmERF061 and ZmMPK6-1 . This parallels findings with ERF6 in Arabidopsis, where MPK3/MPK6-mediated phosphorylation increases protein stability and enhances defense responses against fungal pathogens .

What are the optimal techniques for ERF061 detection using antibodies?

Based on research with other ERF proteins, several techniques can be effectively employed for ERF061 detection:

For immunoblotting specifically, researchers should consider:

• Including proper loading controls (Coomassie blue-stained gels have been effective for other ERF proteins)

• Using appropriate protein extraction buffers that preserve potential phosphorylation states

• Optimizing gel conditions to resolve potential phosphorylated forms, which typically appear as band shifts

What are the best practices for sample preparation when working with ERF061 antibodies?

Effective sample preparation is critical for successful ERF061 antibody applications:

• Protein extraction protocols: For transcription factors like ERF061, nuclear extraction protocols often yield better results. Buffer composition should include protease inhibitors and, importantly, phosphatase inhibitors if studying phosphorylation events .

• Tissue selection and timing: Since ERF061 expression is induced by pathogen treatment and hormone applications, careful timing of tissue collection is essential. For maize, significant ZmERF061 induction was observed after Exserohilum turcicum inoculation .

• Subcellular fractionation: Given ERF061's role as a transcription factor, separating nuclear and cytoplasmic fractions can provide insights into its activation state and translocation dynamics.

• Denaturation conditions: Standard SDS-PAGE conditions are generally suitable, but native conditions may be necessary when studying DNA-binding capabilities.

• Protein stabilization: Based on findings with ERF6, phosphorylation may increase protein stability , so including phosphatase inhibitors is crucial for accurate quantification.

How can researchers distinguish between phosphorylated and non-phosphorylated forms of ERF061?

Distinguishing between phosphorylated and non-phosphorylated forms is critical for understanding ERF061 regulation and requires specific approaches:

• Band shift analysis: Phosphorylation typically causes slower migration on SDS-PAGE. Research on ERF6 demonstrated that phosphorylation at Ser-Pro sites caused detectable upshifts in protein bands visible by immunoblotting .

• Phosphatase treatment controls: Treating protein samples with phosphatases prior to electrophoresis can confirm phosphorylation-dependent mobility shifts. This was effectively used to verify ERF6 phosphorylation .

• Phospho-specific antibodies: Though not specifically mentioned in the search results for ERF061, phospho-specific antibodies that recognize particular phosphorylated residues would be valuable, especially if designed against conserved MAPK target sites (Ser-Pro motifs).

• Mutational analysis approach: Creating versions of ERF061 with mutated potential phosphorylation sites (Ser/Thr to Ala substitutions) can help confirm specific residues that undergo phosphorylation. For ERF6, mutation of Ser-266/Ser-269 residues (ERF6 4A) abolished the phosphorylation-dependent band upshift .

This table based on ERF6 research illustrates how similar mutational approaches could be applied to ERF061 to identify critical phosphorylation sites.

What challenges exist in developing specific antibodies for ERF061 considering the conservation within the ERF family?

Developing highly specific antibodies for ERF061 presents several challenges:

• Structural characteristics: ERF proteins have a unique structural profile where the DNA-binding domains (DBDs) are primarily ordered while the transcriptional regulatory domains (TRDs) are intrinsically disordered . This structural dichotomy affects epitope accessibility and antibody development strategies.

• Domain conservation: The AP2/ERF domain is highly conserved across family members, which can lead to cross-reactivity. Analysis of ERF family proteins shows that the AP2 domains typically have only 0-40% intrinsically disordered amino acids, compared to 40-100% in non-AP2 regions .

• Sequence variability outside conserved domains: While helpful for specificity, the high variability in non-AP2 regions complicates phylogenetic classification and potentially antibody cross-reactivity prediction .

• Protein disorder properties: The intrinsically disordered nature of TRDs in ERF proteins may affect epitope stability and recognition. These regions show compositional profiles typical of intrinsically disordered proteins (IDPs), with an enrichment in disorder-promoting residues .

To address these challenges, researchers should:

• Target unique sequences in the variable regions outside the AP2 domain

• Perform extensive validation against related ERF proteins

• Consider developing antibodies against specific post-translationally modified forms

• Use computational disorder analysis to identify optimal epitope regions

How can ERF061 antibodies be employed to investigate protein-protein interactions, particularly with MPK6?

ERF061 antibodies are valuable tools for investigating critical protein-protein interactions:

• Co-immunoprecipitation (Co-IP): ERF061 antibodies can effectively pull down the protein along with interacting partners. This is particularly relevant for studying the interaction between ZmERF061 and ZmMPK6-1, which has been reported .

• Sequential purification approaches: For cleaner results, sequential purification strategies can be employed. Similar to techniques used with ERF6, this might involve His-tag column purification followed by anti-FLAG antibody affinity gel purification .

• Phosphorylation-dependent interaction studies: Since MPK6 is known to phosphorylate some ERF proteins (like ERF6) , researchers can:

  • Activate the MAPK cascade through pathogen treatment

  • Use Co-IP to detect enhanced association following activation

  • Employ phosphorylation-specific antibodies to confirm modification status

• In vitro kinase assays: Antibodies can be used to purify ERF061 for subsequent in vitro kinase assays with activated MPK6, similar to how activated recombinant MPK3 and MPK6 were shown to phosphorylate ERF6 .

• Transgenic approaches: For in vivo studies, researchers can develop transgenic plants expressing tagged versions of ERF061 (like the 4myc tag used with ERF6) for reliable antibody detection in interaction studies.

What considerations should be made when using ERF061 antibodies across different plant species?

When employing ERF061 antibodies across different plant species, researchers should consider:

Computational analysis of ERF proteins from different species has shown similar amino acid compositional profiles typical of intrinsically disordered proteins, particularly when the AP2 domain is omitted . This suggests some conserved structural features that may impact antibody binding across species.

How can ERF061 antibodies contribute to understanding signal transduction networks in plant immunity?

ERF061 antibodies can significantly advance our understanding of plant immunity signal transduction:

• Pathway integration analysis: By tracking ERF061 phosphorylation and protein levels, researchers can map how this transcription factor integrates signals from multiple pathways. In maize, ZmERF061 mutant lines showed altered expression of both SA signaling-related genes (ZmPR1a) and JA signaling-related genes (ZmLox1) after pathogen infection .

• MAPK cascade connections: Antibodies can help establish the direct relationship between MAPK activation and ERF061 function. Similar to ERF6, which is phosphorylated by MPK3/MPK6 in response to Botrytis cinerea infection , ERF061 likely serves as a downstream component of MAPK signaling.

• Temporal dynamics studies: Antibodies enable precise tracking of ERF061 protein accumulation, modification, and degradation over time following pathogen challenge, revealing the kinetics of the defense response.

• Hormone crosstalk investigation: Since ZmERF061 expression is induced by both SA and MeJA treatments , antibodies can help determine how these hormone pathways converge at the level of protein regulation.

• Functional specificity determination: Unlike some ERF proteins (such as ERF1) whose function depends on ethylene, ZmERF061 appears to function through different regulatory mechanisms , which can be further elucidated using specific antibodies.

What approaches can be used to develop antibodies with custom specificity profiles for ERF061?

Developing antibodies with custom specificity profiles for ERF061 could benefit from approaches similar to those described for other antibody development:

• Biophysics-informed modeling: Researchers can use computational approaches that associate distinct binding modes with different potential epitopes to predict and generate specific antibody variants not observed in initial experimental screens .

• High-throughput sequencing: Selection experiments combined with computational analysis can help identify antibody sequences with desired specificity profiles, either highly specific for ERF061 or with controlled cross-reactivity to related ERF proteins .

• Mode-specific targeting: By identifying different binding modes associated with specific regions of ERF061, researchers can design antibodies that target unique features of the protein .

• Experimental validation pipelines: Custom specificity profiles should be verified through experimental validation, testing antibody variants against various ERF proteins to confirm the desired specificity profile .

The combined approach of biophysics-informed modeling and extensive selection experiments has broad applicability beyond antibodies and offers a powerful toolset for designing proteins with desired physical properties , which could be adapted for ERF061 antibody development.

What are common issues when using ERF061 antibodies and how can they be addressed?

Based on experiences with other ERF proteins, researchers may encounter these challenges when working with ERF061 antibodies:

• Cross-reactivity with related ERF proteins:

  • Solution: Perform validation with recombinant proteins of multiple ERF family members

  • Include appropriate knockout/mutant controls

  • Consider using tagged recombinant proteins for validation

• Poor detection of phosphorylated forms:

  • Solution: Ensure phosphatase inhibitors are included in extraction buffers

  • Optimize gel conditions to resolve phosphorylated and non-phosphorylated forms

  • Consider phosphatase treatment controls to confirm band identity

• Insufficient nuclear extraction:

  • Solution: Use optimized nuclear extraction protocols specific for transcription factors

  • Verify extraction efficiency with known nuclear markers

• Low signal due to protein instability:

  • Solution: Based on ERF6 research, phosphorylation may increase protein stability

  • Consider using proteasome inhibitors if studying protein degradation dynamics

  • Optimize protein extraction conditions to preserve both modified and unmodified forms

• Interference from intrinsically disordered regions:

  • Solution: Target antibodies to regions with higher structural stability

  • Use computational disorder analysis to identify optimal epitope regions

  • Consider multiple antibodies targeting different regions of the protein

Addressing these challenges requires careful experimental design and appropriate controls to ensure reliable and reproducible results when working with ERF061 antibodies.