ERF070 Antibody

What is ERF070 Antibody and what are its primary research applications?

ERF070 Antibody is a research-grade antibody that recognizes epitopes of the ERF070 protein, which functions as a transcription factor in plants, particularly in Arabidopsis thaliana (Mouse-ear cress). The antibody is primarily used in plant molecular biology research to study transcriptional regulation, stress responses, and developmental processes. As with other antibodies in the ERF (Ethylene Response Factor) family, ERF070 Antibody enables researchers to investigate signaling pathways related to plant hormone responses, particularly ethylene signaling cascades .

The applications of this antibody include Western blotting, immunoprecipitation, chromatin immunoprecipitation (ChIP), and immunohistochemistry in plant tissues. Understanding its specificity and optimal working conditions is essential for accurate experimental outcomes in plant biology research.

How should ERF070 Antibody be stored and handled to maintain optimal activity?

Proper storage and handling of ERF070 Antibody are crucial to preserve its activity and specificity. The antibody should be stored at -20°C for long-term preservation, with aliquoting recommended to avoid repeated freeze-thaw cycles that can degrade antibody quality. When working with the antibody, researchers should maintain cold chain protocols, keeping it on ice during experiments.

For daily use, small aliquots can be stored at 4°C for up to two weeks. The antibody is typically shipped with stabilizers and preservatives that help maintain its integrity during transport and storage. Always follow manufacturer-specific recommendations, as formulation details may vary between suppliers. Proper handling ensures consistent experimental results and extends the usable lifetime of this research resource .

What validation methods confirm ERF070 Antibody specificity for experimental applications?

Validation of ERF070 Antibody specificity is essential before using it in critical experiments. Multiple validation methods should be employed, including:

• Western blot analysis with positive controls (expressing ERF070) and negative controls (tissues or cells known not to express the target)

• Peptide competition assays to confirm binding to the intended epitope

• Knockout/knockdown validation using CRISPR-Cas9 or RNAi techniques

• Cross-reactivity testing against related ERF family proteins to ensure specificity

• Immunoprecipitation followed by mass spectrometry to confirm target capture

Researchers should note that antibody validation is context-dependent, and an antibody that works well for Western blotting may not perform optimally for immunohistochemistry. Therefore, validation should be performed for each specific application. Documentation of validation experiments is vital for research reproducibility and should be maintained in laboratory records .

How can ERF070 Antibody be utilized in chromatin immunoprecipitation sequencing (ChIP-seq) to identify transcription factor binding sites?

ERF070 Antibody can be employed in ChIP-seq experiments to map genome-wide binding sites of the ERF070 transcription factor, providing insights into its regulatory networks. The optimization process for ChIP-seq with ERF070 Antibody involves several critical steps:

First, crosslinking conditions must be optimized specifically for plant tissues, typically using 1% formaldehyde for 10-15 minutes at room temperature. The chromatin fragmentation protocol requires careful calibration, with sonication parameters adjusted to achieve fragments of 200-500 bp for optimal resolution.

For immunoprecipitation, antibody concentration needs titration to determine the optimal amount (typically 2-5 μg per reaction). Including appropriate controls is essential: IgG negative controls and input chromatin for normalization. Following immunoprecipitation, thorough washing steps are necessary to reduce background, and elution conditions should be optimized to maximize recovery of bound DNA.

The ChIP-seq library preparation should follow standard protocols with adjustments for potentially limited DNA yield from plant samples. Data analysis requires specialized bioinformatics pipelines to identify binding motifs and correlate with gene expression data. This approach allows researchers to construct comprehensive regulatory networks involving ERF070 in response to various stimuli or developmental stages .

What are the considerations for developing a multiplexed assay including ERF070 Antibody with other antibodies targeting related transcription factors?

Developing multiplexed assays that include ERF070 Antibody alongside antibodies targeting related transcription factors requires careful consideration of several technical factors:

• Antibody compatibility: Each antibody must be raised in different host species to allow for species-specific secondary detection. If using antibodies from the same host, direct conjugation to different fluorophores is necessary.

• Epitope accessibility: Consider whether the binding of one antibody might sterically hinder the binding of another, particularly if targeting proteins within the same complex.

• Signal separation: When using fluorescent detection, select fluorophores with minimal spectral overlap or implement appropriate compensation controls.

• Cross-reactivity assessment: Comprehensive testing for cross-reactivity between antibodies and non-target proteins is essential before multiplexing.

• Sequential staining protocols: For challenging combinations, sequential staining with complete stripping between steps may be necessary.

A successful multiplexed assay allows researchers to examine the cooperative or competitive binding patterns of multiple ERF family transcription factors at regulatory regions, providing insights into complex transcriptional networks governing plant stress responses and development. This approach is particularly valuable when studying signaling pathway crosstalk and redundancy within the ERF family .

How can ERF070 Antibody be employed in proximity-dependent biotin identification (BioID) studies to map protein interaction networks?

Implementing ERF070 Antibody in BioID studies represents an advanced application for mapping the protein interactome of ERF070 transcription factor. This approach requires several methodological considerations:

First, researchers must generate fusion constructs combining ERF070 with a BioID2 or TurboID enzyme under a suitable promoter for expression in plant systems. Following transformation and expression verification, biotin labeling is performed by supplementing growth media with biotin (typically 50 μM) for 12-24 hours.

After labeling, tissues are harvested and lysed under denaturing conditions to solubilize biotinylated proteins. ERF070 Antibody can be used in parallel immunoprecipitation experiments to validate the expression and localization of the fusion protein. Biotinylated proteins are captured using streptavidin beads, followed by stringent washing to remove non-specific interactions.

The captured proteins are then identified through mass spectrometry analysis. Data interpretation requires careful filtering against appropriate controls to distinguish true interactors from background proteins. This methodology allows researchers to discover novel protein-protein interactions in the ERF070 signaling network, including transient interactions that might be missed by conventional co-immunoprecipitation approaches .

What controls should be included when using ERF070 Antibody in immunoprecipitation experiments?

When designing immunoprecipitation (IP) experiments with ERF070 Antibody, a comprehensive set of controls is essential to ensure valid and interpretable results:

• Input control: A small portion (5-10%) of the pre-cleared lysate should be reserved before immunoprecipitation to verify the presence of the target protein and allow quantitative comparison.

• Isotype control: An irrelevant antibody of the same isotype and host species as the ERF070 Antibody should be used in parallel reactions to identify non-specific binding.

• No-antibody control: Running the IP procedure without any antibody helps identify proteins that bind non-specifically to the beads or support matrix.

• Knockout/knockdown control: When available, samples from ERF070 knockout or knockdown lines provide the most stringent negative control.

• Peptide competition control: Pre-incubating the ERF070 Antibody with excess immunizing peptide before IP can confirm binding specificity.

• Reciprocal IP: If investigating protein-protein interactions, confirming interactions by immunoprecipitating with antibodies against the putative interacting partner.

Proper implementation of these controls allows researchers to confidently distinguish specific from non-specific interactions and accurately interpret immunoprecipitation results. The controls should be processed identically to the experimental samples throughout the entire protocol to ensure valid comparisons .

What are the optimal fixation and antigen retrieval methods when using ERF070 Antibody for immunohistochemistry in plant tissues?

Optimizing fixation and antigen retrieval for ERF070 Antibody in plant immunohistochemistry requires methodical testing of multiple parameters:

For fixation, paraformaldehyde-based protocols (3-4% PFA) typically yield better results than glutaraldehyde, which can cause excessive cross-linking and epitope masking. The optimal fixation duration varies with tissue type: 2-4 hours for thin sections and leaves, overnight for thicker tissues and roots. Vacuum infiltration during fixation significantly improves penetration through plant cell walls and cuticles.

Antigen retrieval represents a critical step that often determines success or failure in plant immunohistochemistry. Heat-induced epitope retrieval using citrate buffer (pH 6.0) at 95°C for 20-30 minutes frequently provides good results with ERF070 Antibody. For tissues with high phenolic content, adding 0.1% Triton X-100 to the retrieval buffer can improve accessibility.

Enzymatic antigen retrieval using a combination of cell wall-degrading enzymes (1% cellulase, 0.5% macerozyme, 0.1% pectolyase) at room temperature for 30-60 minutes sometimes achieves better results than heat-based methods for nuclear transcription factors like ERF070.

Permeabilization steps should be carefully optimized, as excessive detergent can disrupt nuclear architecture while insufficient permeabilization prevents antibody access. A gradual approach starting with 0.1% Triton X-100 and titrating upward if necessary is recommended .

How should epitope masking be addressed when using ERF070 Antibody to study protein complexes?

Epitope masking presents a significant challenge when using ERF070 Antibody to study protein complexes, particularly when the antibody's target epitope might be obscured by protein-protein interactions. Several strategies can address this limitation:

• Alternative antibody selection: Utilizing multiple antibodies targeting different epitopes of ERF070 increases the probability of successful detection regardless of complex formation. Combining N-terminal and C-terminal targeting antibodies provides complementary information.

• Gentle detergent treatment: Implementing mild detergents (0.1% NP-40 or 0.5% Triton X-100) can partially disrupt protein-protein interactions without denaturing the target protein, potentially exposing masked epitopes.

• Cross-linking optimization: When studying transient interactions, titrating cross-linker concentration and incubation time helps balance between capturing interactions and maintaining epitope accessibility.

• Denaturing vs. native conditions: Comparing results obtained under native versus denaturing conditions can reveal masked epitopes and provide insights into complex formation.

• Proximity labeling approaches: Complementing traditional antibody-based detection with proximity labeling techniques (BioID or APEX) can overcome epitope masking limitations.

• Competitive elution strategies: Using peptides corresponding to known interaction interfaces to competitively disrupt specific protein-protein interactions can free masked epitopes.

By implementing these approaches systematically, researchers can distinguish between true negative results and false negatives caused by epitope masking, leading to more comprehensive characterization of ERF070-containing protein complexes .

What strategies can address non-specific binding when using ERF070 Antibody in Western blot applications?

Non-specific binding in Western blot applications with ERF070 Antibody can significantly complicate data interpretation. A systematic troubleshooting approach involves:

• Blocking optimization: Test different blocking agents beyond the standard 5% non-fat dry milk, including 5% BSA, commercial blocking buffers, or a combination of gelatin and casein. The optimal blocking agent varies depending on the specific antibody and sample type.

• Antibody dilution titration: Perform a dilution series (typically ranging from 1:500 to 1:5000) to identify the optimal concentration that maximizes specific signal while minimizing background.

• Buffer modifications: Adjusting the ionic strength of wash buffers by increasing NaCl concentration (from 150mM to 300-500mM) can disrupt weak, non-specific interactions. Adding 0.1-0.5% SDS to TBST or PBST wash buffers can further reduce non-specific binding.

• Extended washing protocols: Implementing additional washing steps (6-8 washes of 10 minutes each) with fresh buffer each time significantly reduces background.

• Pre-adsorption: For particularly problematic antibodies, pre-adsorbing with plant extracts from tissues not expressing the target protein can sequester antibodies that bind non-specifically.

• Two-dimensional electrophoresis: If multiple bands persist, 2D-PAGE can help distinguish between true isoforms and non-specific binding based on both molecular weight and isoelectric point.

• Alternative detection systems: Switching from chemiluminescent to fluorescent secondary antibodies often provides cleaner results with lower background.

Implementing these strategies systematically, changing only one parameter at a time, allows researchers to optimize Western blot protocols specifically for ERF070 Antibody .

How can researchers troubleshoot weak or absent signals when using ERF070 Antibody in immunohistochemistry?

When troubleshooting weak or absent signals in immunohistochemistry experiments with ERF070 Antibody, researchers should systematically investigate multiple aspects of the protocol:

• Sample preparation assessment: Inadequate fixation can lead to antigen loss, while excessive fixation causes epitope masking. Testing a gradient of fixation times (1 hour to overnight) can identify optimal conditions. For plant tissues, ensure proper tissue penetration using vacuum infiltration during fixation.

• Antigen retrieval optimization: Nuclear transcription factors like ERF070 often require aggressive antigen retrieval. Test multiple methods in parallel:

  • Heat-induced epitope retrieval with citrate buffer (pH 6.0)

  • Tris-EDTA buffer (pH 9.0) for 20-30 minutes at 95°C

  • Enzymatic digestion with plant-specific cell wall degrading enzymes

• Antibody concentration adjustment: Primary antibody may need higher concentrations for immunohistochemistry than for Western blotting. Try a concentration series from 1:50 to 1:500.

• Incubation conditions modification: Extended primary antibody incubation (overnight at 4°C or even 48-72 hours for thick plant sections) can significantly improve signal.

• Detection system amplification: Consider signal amplification methods such as:

  • Tyramide signal amplification (TSA) which can increase sensitivity 10-100 fold

  • Poly-HRP detection systems

  • Multiple layers of biotinylated secondary antibodies

• Detergent optimization: Nuclear proteins require sufficient permeabilization. Test increasing concentrations of Triton X-100 (0.1-3%) or substitute with saponin (0.1-0.5%) which creates smaller pores in membranes.

• Positive control implementation: Process known positive control samples alongside experimental samples to distinguish between technical issues and biological absence of the target .

What approaches can resolve inconsistent results between different lots of ERF070 Antibody?

Lot-to-lot variability is a common challenge when working with research antibodies like ERF070 Antibody. Resolving inconsistencies requires a multi-faceted approach:

• Batch validation protocol: Implement a standardized validation protocol for each new antibody lot, including:

  • Western blot against known positive controls

  • Immunoprecipitation efficiency testing

  • Immunohistochemistry on reference samples

• Reference sample banking: Maintain frozen aliquots of reference samples (protein extracts, fixed tissues) that have worked well with previous antibody lots as benchmarks for testing new lots.

• Epitope mapping: If significant variability occurs, consider epitope mapping to determine if different lots recognize distinct epitopes within the ERF070 protein. This can be accomplished through:

  • Peptide arrays covering the entire protein sequence

  • Truncation mutants expressing different segments of the protein

  • Competition assays with synthetic peptides

• Validation documentation: Implement comprehensive documentation of antibody performance metrics including:

  • Signal-to-noise ratio under standardized conditions

  • Minimum detectable concentration

  • Cross-reactivity profile

  • Optimal working dilutions for each application

• Supplier communication: Engage with antibody suppliers about observed inconsistencies, as they may have internal quality control data that can explain variability or offer replacement lots.

• Pooling strategy: For critical experiments, consider purchasing multiple antibody vials from the same lot to ensure consistency throughout a project lifecycle.

By implementing these strategies, researchers can minimize the impact of lot-to-lot variability and maintain experimental consistency when working with ERF070 Antibody over extended research timelines .

How should researchers analyze and quantify Western blot data generated using ERF070 Antibody for accurate protein expression comparisons?

Accurate quantification of Western blot data using ERF070 Antibody requires rigorous analytical approaches to ensure reliable protein expression comparisons:

• Image acquisition optimization: Capture images using a digital imaging system with a linear dynamic range (16-bit CCD camera) rather than film, which has a limited linear range. Ensure exposure settings avoid pixel saturation by checking histogram data during image capture.

• Normalization strategy selection: Select appropriate loading controls based on experimental context. For transcription factors like ERF070, nuclear loading controls (Histone H3) are often more appropriate than cytoplasmic housekeeping proteins (GAPDH, β-actin).

• Densitometry methodology: Utilize specialized software (ImageJ, Image Lab, etc.) to perform densitometry. Define signal boundaries consistently across all lanes and subtract local background values rather than global background.

• Technical replicate processing: Analyze three independent biological replicates with technical duplicates. Calculate coefficient of variation (CV) between technical replicates; values exceeding 15% warrant further optimization.

• Statistical analysis application: Apply appropriate statistical tests based on experimental design. For multiple condition comparisons, use ANOVA with post-hoc tests rather than multiple t-tests to control familywise error rates.

• Dose-response validation: When studying induction or inhibition of ERF070 expression, perform dose-response experiments to confirm biological relevance and rule out non-specific effects.

The table below illustrates recommended quantification parameters for Western blot analysis:

This systematic approach enables reliable quantitative comparisons of ERF070 protein levels across experimental conditions .

What analytical approaches are recommended for interpreting chromatin immunoprecipitation data generated with ERF070 Antibody?

Interpreting chromatin immunoprecipitation (ChIP) data generated with ERF070 Antibody requires specialized analytical approaches to extract meaningful biological insights:

• Quality control metrics: Begin by assessing quality control parameters including:

  • Enrichment of positive control regions (known ERF070 binding sites) versus negative control regions

  • Signal-to-noise ratio calculation for each experiment

  • Fragment size distribution analysis to confirm proper chromatin shearing

  • Library complexity evaluation to ensure adequate sequencing depth

• Peak calling optimization: Select appropriate peak calling algorithms (MACS2, GEM, HOMER) with parameters optimized for transcription factors. ERF070 typically displays sharp binding peaks, requiring different settings than histone modification analysis.

• Differential binding analysis: For comparative studies, implement specialized differential binding analysis tools (DiffBind, MAnorm) with proper normalization for sequencing depth and global binding differences.

• Motif enrichment analysis: Perform de novo motif discovery using MEME, HOMER, or similar tools to identify ERF070 binding motifs. Compare discovered motifs with known ERF family binding preferences to identify unique and shared recognition elements.

• Genomic feature association: Analyze the distribution of binding sites relative to genomic features (promoters, enhancers, gene bodies) using tools like ChIPseeker or GREAT. Calculate enrichment statistics compared to genomic background.

• Integration with transcriptomic data: Correlate binding sites with differential gene expression data to identify direct regulatory targets versus indirect effects. Categorize genes based on presence/absence of binding and direction of expression change.

• Pathway and gene ontology analysis: Apply pathway enrichment analysis to ERF070-bound genes to identify biological processes under its regulation. Compare enriched terms under different experimental conditions to detect context-specific functions.

• Visualization strategies: Generate composite plots showing average binding profiles around features of interest (TSS, TTS, enhancers) and heatmaps displaying binding intensity across experimental conditions for identified target genes.

This comprehensive analytical framework enables researchers to translate ChIP-seq data into mechanistic insights about ERF070's role in transcriptional regulation networks .

How can researchers distinguish between direct and indirect effects when studying ERF070 function using antibody-based approaches?

Distinguishing between direct and indirect effects when studying ERF070 function requires integrated experimental approaches and careful data interpretation:

• Temporal analysis implementation: Conduct time-course experiments following ERF070 induction or inhibition. Direct targets typically show more rapid response (within 0.5-2 hours) while indirect targets show delayed responses (4+ hours). This temporal separation helps classify primary versus secondary effects.

• Pharmacological intervention: Utilize protein synthesis inhibitors (cycloheximide) to block translation of intermediate regulatory proteins. Genes that remain responsive to ERF070 modulation even in the presence of cycloheximide are likely direct targets.

• Binding site correlation: Integrate ChIP-seq data with expression data, categorizing genes as:

  • Bound and regulated (high confidence direct targets)

  • Bound but not regulated (potential condition-specific targets)

  • Regulated but not bound (likely indirect targets)

  • Neither bound nor regulated (unrelated genes)

• Motif presence analysis: Examine promoters of regulated genes for ERF070 binding motifs. Direct targets typically contain consensus binding sites, while indirect targets may lack these motifs.

• Inducible systems utilization: Employ rapid induction systems (e.g., dexamethasone-inducible or estradiol-inducible ERF070) to trigger ERF070 activity with precise temporal control, allowing separation of primary and secondary responses.

• Genome editing validation: Perform targeted mutation of putative ERF070 binding sites using CRISPR-Cas9 to confirm the functional relevance of specific binding events. Loss of regulation following binding site mutation provides strong evidence for direct regulation.

• Network modeling application: Develop mathematical models of the regulatory network that incorporate both direct and indirect interactions. Test model predictions against experimental data to refine understanding of network architecture.

These integrated approaches allow researchers to construct high-confidence regulatory networks distinguishing direct ERF070 targets from downstream effectors, providing deeper insight into the protein's biological function .