ERF7 Antibody

What is ERF7 and how does it relate to other ERF family members?

ERF7 (Ethylene Response Factor 7) belongs to the ERF/AP2 transcription factor family, which plays crucial roles in plant stress responses. ERF proteins contain the AP2 DNA-binding domain and are classified into various subfamilies. The ERF-VII subfamily includes key members like RAP2.2, RAP2.12, RAP2.3, HRE1 (ERF73), and HRE2 that regulate hypoxia-responsive genes . While our search results do not specifically detail ERF7, based on the ERF family characteristics, it likely functions as a transcription factor involved in ethylene signaling pathways and stress responses similar to other family members such as ERF71 and ERF73 .

What are the primary applications of ERF7 antibodies in plant research?

ERF7 antibodies are valuable tools for:

• Protein detection via Western blotting

• Protein localization using immunohistochemistry

• Chromatin immunoprecipitation (ChIP) assays to identify DNA binding sites

• Protein-protein interaction studies

The methodological approach for these applications would be similar to those used with other ERF family antibodies, such as the chromatin immunopurification techniques used with RAP2.2 and RAP2.12 to study binding to hypoxia-responsive promoter elements (HRPEs) .

What validated control samples should be included when using ERF7 antibodies?

For experimental validity when using ERF7 antibodies, researchers should include:

Similar controls were employed in ERF-VII transcription factor research, where researchers used wild-type Col-0 as negative controls when analyzing FLAG-tagged RAP2.2 or RAP2.12 binding to promoter regions .

How can ERF7 antibodies be utilized to investigate transcriptional regulation mechanisms?

ERF7 antibodies can be employed in chromatin immunoprecipitation (ChIP) experiments to identify genomic regions bound by ERF7. This approach would be similar to the ChIP methodology used for RAP2.2 and RAP2.12, which demonstrated binding to specific promoter regions containing hypoxia-responsive promoter elements (HRPEs) .

The procedure would involve:

• Cross-linking proteins to DNA in plant tissues

• Shearing chromatin to 200-400bp fragments

• Immunoprecipitating with ERF7 antibody

• Purifying and analyzing co-precipitated DNA by qPCR or sequencing

• Identifying ERF7 binding sites based on enrichment patterns

For robust results, researchers should include appropriate controls such as wild-type samples without tagged proteins, as demonstrated in the FLAG-RAP2.2 and FLAG-RAP2.12 ChIP experiments .

What approaches should be used to validate ERF7 antibody specificity?

Validating antibody specificity is critical for meaningful research outcomes. For ERF7 antibodies, consider:

• Western blot analysis using:

  • Recombinant ERF7 protein

  • Plant tissues with ERF7 overexpression

  • erf7 mutant tissues (should show no signal)

  • Competition assays with immunizing peptide

• Cross-reactivity assessment:

  • Testing against closely related ERF proteins like other subfamily members

  • Performing immunoprecipitation followed by mass spectrometry

• Genetic validation:

  • Comparing antibody signals between wild-type and erf7 knockout lines

  • Using complementation lines to verify restored detection

This approach aligns with methods that would be used to validate other ERF antibodies, such as those against ERF71 or ERF73 .

How can ERF7 antibodies be used to investigate protein-protein interactions within transcriptional complexes?

To investigate ERF7's role in transcriptional complexes:

• Co-immunoprecipitation (Co-IP):

  • Use ERF7 antibodies to pull down ERF7 and associated proteins

  • Analyze by mass spectrometry to identify interaction partners

  • Confirm interactions using reverse Co-IP with antibodies against candidate partners

• Proximity labeling approaches:

  • Express ERF7 fused to BioID or APEX2

  • Identify proximal proteins through biotinylation and streptavidin pulldown

  • Validate candidates using ERF7 antibodies

• Bimolecular fluorescence complementation:

  • Express ERF7 fused to split fluorescent protein fragment

  • Co-express candidate interactors fused to complementary fragment

  • Validate interactions observed through fluorescence using ERF7 antibodies

Similar approaches would be applicable to studying protein interactions of other ERF family members like RAP2.2 and RAP2.12, which were shown to function redundantly in hypoxia response regulation .

What are the optimal sample preparation conditions for Western blot detection of ERF7?

For optimal ERF7 detection by Western blot:

• Sample preparation:

  • Extract proteins in buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100

  • Include protease inhibitors (PMSF, leupeptin, pepstatin)

  • Add phosphatase inhibitors if phosphorylation status is important

  • For nuclear proteins like ERF7, use nuclear extraction protocols

• Gel electrophoresis parameters:

  • Use 10-12% SDS-PAGE gels

  • Load 20-40μg total protein per lane

  • Include molecular weight markers

• Transfer and detection:

  • Transfer to PVDF membrane (better for nuclear proteins)

  • Block with 5% non-fat milk or BSA

  • Use ERF7 antibody at optimized dilution (typically 1:1000)

  • Include controls as outlined in section 1.3

These recommendations are based on general practices for transcription factor detection and would be similar to those used for other ERF family proteins such as ERF71 and ERF73 .

What strategies can overcome cross-reactivity issues when using ERF7 antibodies?

When facing cross-reactivity issues with ERF7 antibodies:

• Antibody purification techniques:

  • Perform affinity purification against specific ERF7 epitopes

  • Use competitive elution with immunizing peptide

  • Consider cross-adsorption against related ERF proteins

• Experimental modifications:

  • Increase stringency of washing steps (higher salt concentration)

  • Optimize antibody concentration (use titration experiments)

  • Adjust blocking conditions (try different blocking agents)

• Alternative validation approaches:

  • Compare results with orthogonal detection methods

  • Use tagged ERF7 constructs in parallel experiments

  • Employ genetic controls (erf7 mutants, complementation lines)

When selecting antibodies, researchers should carefully evaluate specificity information provided by suppliers, similar to what's shown for ERF71 and ERF73 antibodies in their product information .

How can ERF7 antibodies be effectively used to study post-translational modifications?

To investigate post-translational modifications (PTMs) of ERF7:

• Modification-specific detection:

  • Use phospho-specific antibodies if available

  • Combine ERF7 antibodies with PTM-specific antibodies

  • Employ 2D gel electrophoresis to separate modified forms

• Enrichment approaches:

  • Immunoprecipitate with ERF7 antibodies followed by PTM detection

  • Use PTM-specific enrichment (e.g., phospho-peptide enrichment) before ERF7 detection

  • Perform sequential immunoprecipitation with ERF7 and PTM antibodies

• Analytical techniques:

  • Mass spectrometry analysis after ERF7 immunoprecipitation

  • Use mobility shift assays to detect modified forms

  • Employ Phos-tag gels for phosphorylated protein separation

This approach would be particularly relevant for studying ERF7, as ERF-VII family proteins are known to undergo regulated proteolysis via the N-end rule pathway in an oxygen-dependent manner, similar to what has been observed with RAP2.2 and RAP2.12 .

How can ERF7 antibodies be incorporated into ChIP-seq workflows to identify genome-wide binding sites?

For ChIP-seq using ERF7 antibodies:

• Experimental setup:

  • Cross-link proteins to DNA (1% formaldehyde, 10 minutes)

  • Sonicate chromatin to 200-300bp fragments

  • Immunoprecipitate using ERF7 antibodies

  • Prepare sequencing libraries from immunoprecipitated DNA

  • Sequence using next-generation sequencing platforms

• Data analysis pipeline:

  • Align reads to reference genome

  • Call peaks using MACS2 or similar software

  • Perform motif discovery on peak regions

  • Integrate with transcriptome data to identify regulated genes

• Validation strategies:

  • Confirm selected binding sites using ChIP-qPCR

  • Perform reporter assays for identified promoters

  • Correlate binding with gene expression changes

This methodology would be similar to the approach used to identify the hypoxia-responsive promoter element (HRPE) bound by RAP2.2 and RAP2.12 in hypoxia-responsive genes, as reported in search result .

What considerations should guide experimental design when using ERF7 antibodies to study stress responses?

When designing experiments to study ERF7's role in stress responses:

• Stress treatment design:

  • Include appropriate time course (early and late responses)

  • Use physiologically relevant stress conditions

  • Monitor stress markers to confirm treatment efficacy

• Sampling considerations:

  • Collect tissue-specific samples (roots, leaves, etc.)

  • Consider developmental stage effects

  • Use flash-freezing to preserve protein modifications

• Comparative analysis:

  • Include related ERF family members for comparison

  • Examine different stress types to assess specificity

  • Study interactions with key signaling components

These considerations would be particularly relevant as ERF family members like RAP2.2, RAP2.12, and HRE1/HRE2 have been shown to respond differentially to hypoxia and other stress conditions .

How can ERF7 antibodies be used alongside CRISPR-Cas9 gene editing to validate functional studies?

Integrating ERF7 antibodies with CRISPR-Cas9 approaches:

• Validation of knockout/knockdown:

  • Use ERF7 antibodies to confirm protein absence in CRISPR-edited lines

  • Quantify reduction in protein levels in partial knockdowns

  • Verify specificity by confirming presence of other ERF proteins

• Domain function analysis:

  • Generate domain-specific mutations using CRISPR-Cas9

  • Use ERF7 antibodies to confirm stable protein expression

  • Compare binding patterns of wild-type vs. mutant proteins

• Complementation studies:

  • Reintroduce wild-type or mutant ERF7 into knockout backgrounds

  • Use antibodies to confirm expression levels

  • Compare functional recovery with protein expression

This integrated approach would be similar to the techniques used to study the redundant functions of RAP2.2 and RAP2.12 through mutant analysis and complementation .

How should researchers interpret conflicting results between ERF7 antibody-based detection and transcript levels?

When faced with discrepancies between protein and mRNA data:

• Technical considerations:

  • Verify antibody specificity and sensitivity

  • Confirm RNA analysis methods and normalization

  • Check for technical issues in either workflow

• Biological explanations:

  • Consider post-transcriptional regulation mechanisms

  • Evaluate protein stability and turnover rates

  • Assess tissue-specific expression patterns

• Integrated analysis approach:

  • Use multiple detection methods to validate results

  • Perform time-course experiments to capture dynamics

  • Consider subcellular fractionation to assess protein localization

This analytical approach is particularly relevant for ERF family proteins, as research on ERF-VII members has shown that post-translational regulation via the N-end rule pathway plays a crucial role in their function, with protein stability being regulated by oxygen levels independently of transcript abundance .

What statistical approaches are most appropriate for analyzing ChIP data generated using ERF7 antibodies?

For robust statistical analysis of ERF7 ChIP data:

• Peak calling statistics:

  • Use false discovery rate (FDR) < 0.05 for peak identification

  • Apply fold-enrichment thresholds (typically >2-fold over input)

  • Consider replicate consistency (peaks present in multiple biological replicates)

• Comparative analyses:

  • Employ differential binding analysis between conditions

  • Use normalized read counts for quantitative comparisons

  • Apply appropriate transformations for data normalization

• Integration with other data types:

  • Correlate binding strength with gene expression changes

  • Perform gene ontology enrichment on target genes

  • Analyze co-occurrence with other transcription factor binding sites

These statistical approaches align with methodologies that would be used when analyzing ChIP data for other ERF family members, such as the ChIP experiments conducted with FLAG-RAP2.2 and FLAG-RAP2.12 .

How can researchers differentiate between direct and indirect effects when studying ERF7 regulatory networks?

To distinguish direct from indirect regulatory effects:

• Experimental strategies:

  • Combine ChIP-seq with RNA-seq from the same conditions

  • Use inducible expression systems with time-course sampling

  • Perform reporter assays with wild-type and mutated binding sites

• Data integration methods:

  • Identify genes with both binding sites and expression changes

  • Analyze temporal patterns of binding and expression changes

  • Compare effects of ERF7 mutations on binding vs. gene expression

• Network analysis approaches:

  • Build directed regulatory networks

  • Use causal inference algorithms

  • Implement mathematical modeling of regulatory dynamics

This approach would be similar to the methods used to identify direct targets of RAP2.2 and RAP2.12 through the identification of the hypoxia-responsive promoter element and subsequent validation using promoter mutations and transactivation assays .