ERF036 Antibody

What is the target protein of ERF036 Antibody and what is its cellular function?

ERF036 Antibody targets the Ethylene-responsive transcription factor ERF036, which likely functions as a transcriptional activator. The protein belongs to the AP2/ERF transcription factor family, specifically the ERF subfamily. It binds to the GCC-box pathogenesis-related promoter element and plays a critical role in regulating gene expression in response to stress factors and components of stress signal transduction pathways.

The protein is primarily localized in the nucleus, consistent with its role as a transcription factor. It is also known by several synonyms including At3g16280, MYA6.9, and T02O04.22. Understanding the target protein's function is essential for designing appropriate experiments and interpreting results when using this antibody.

What applications is ERF036 Antibody suitable for in research settings?

Based on available information and antibody characteristics, ERF036 Antibody can be used in various research applications, though specific validations should be performed for each application. The antibody is provided in liquid form with a buffer containing 50% Glycerol and 0.01M Phosphate Buffered Saline (PBS) at pH 7.4, with 0.03% Proclin 300 as a preservative.

Potential applications include:

• Western blotting

• Immunohistochemistry (IHC)

• Immunofluorescence

• Immunoprecipitation (IP)

• Chromatin immunoprecipitation (ChIP)

When planning experiments, researchers should consider that antibody validation is critical for ensuring reliable results, as highlighted by research showing that insufficient antibody validation has challenged research in other fields .

How should researchers validate ERF036 Antibody specificity before experimental use?

Proper antibody validation is crucial to ensure experimental reliability. Based on best practices in antibody research, researchers should implement a multi-step validation process:

• Positive and negative controls: Use tissues or cell lines known to express or not express ERF036.

• Western blot analysis: Confirm that the antibody detects a band of the expected molecular weight.

• Knockdown/knockout validation: Test the antibody in samples where the target protein has been depleted through siRNA, CRISPR, or other techniques.

• Immunoprecipitation followed by mass spectrometry: This approach can confirm that the antibody is indeed pulling down the intended target protein .

• Cross-reactivity testing: Assess potential cross-reactivity with similar proteins, particularly other members of the AP2/ERF family.

Recent research has shown that many antibodies lack proper validation, leading to inconsistent results across studies . Therefore, thorough validation before experimental use is essential for generating reliable data with ERF036 Antibody.

How can researchers address potential cross-reactivity of ERF036 Antibody with other AP2/ERF family members?

The AP2/ERF transcription factor family contains multiple members with structural similarities, creating potential for cross-reactivity. Advanced researchers should implement several strategies to ensure specificity:

• Peptide competition assays: Pre-incubating the antibody with the immunizing peptide should abolish specific binding signals.

• Parallel analysis with other AP2/ERF antibodies: Using antibodies against related family members can help identify potential cross-reactivity patterns.

• Recombinant protein analysis: Testing the antibody against a panel of recombinant AP2/ERF family proteins can define the specificity profile.

• Sequential immunoprecipitation: This technique can help identify potential cross-reactive proteins by first depleting the primary target and then analyzing remaining bound proteins.

• Mass spectrometry analysis: Immunoprecipitation followed by mass spectrometry can definitively identify all proteins recognized by the antibody .

The issue of cross-reactivity is particularly relevant given evidence from other fields showing that even broadly accepted antibodies can generate discordant expression patterns .

What are the optimal conditions for using ERF036 Antibody in chromatin immunoprecipitation (ChIP) experiments?

ChIP experiments with ERF036 Antibody require careful optimization due to the nature of transcription factor binding to DNA. While specific protocols for ERF036 are not detailed in the provided search results, best practices based on similar transcription factor antibodies include:

• Fixation optimization: Test different formaldehyde concentrations (0.5-1%) and fixation times (10-20 minutes) to preserve protein-DNA interactions without overfixing.

• Sonication parameters: Optimize sonication conditions to generate DNA fragments of 200-500 bp.

• Antibody concentration: Titrate antibody amounts (typically 2-5 μg per reaction) to determine optimal signal-to-noise ratio.

• Washing stringency: Test different salt concentrations in wash buffers to minimize background while maintaining specific signals.

• Controls: Always include input controls, IgG controls, and if possible, a positive control locus known to be bound by ERF036.

Some transcription factors require dual crosslinking with DSG (disuccinimidyl glutarate) before formaldehyde treatment, which may be worth exploring if standard protocols yield poor results.

How should researchers analyze contradictory data when using ERF036 Antibody across different experimental platforms?

When faced with contradictory results across different applications (e.g., western blot showing one pattern of expression while IHC shows another), researchers should implement a systematic troubleshooting approach:

• Reassess antibody specificity: Perform additional validation experiments as outlined in question 1.3.

• Evaluate sample preparation differences: Different applications require different sample preparation methods that may affect epitope availability.

• Consider protein modifications: Post-translational modifications may impact antibody recognition in different contexts.

• Analyze splice variants: Determine if ERF036 has splice variants that might be differentially detected.

• Implement orthogonal approaches: Use alternative methods to detect the protein, such as RNA expression analysis or CRISPR-based tagging.

Recent research has highlighted that only 1 out of 13 antibodies for a different nuclear receptor was sufficiently specific in IHC applications, despite many being marketed as validated . This emphasizes the importance of rigorous validation across all intended applications.

What considerations should researchers take into account when using ERF036 Antibody for immunohistochemistry?

Immunohistochemistry with ERF036 Antibody requires careful optimization:

• Fixation method: Test both formalin-fixed, paraffin-embedded (FFPE) and frozen section protocols to determine optimal epitope preservation.

• Antigen retrieval: Optimize antigen retrieval methods (heat-induced vs. enzymatic) and buffers (citrate vs. EDTA) to maximize signal while minimizing background.

• Blocking conditions: Test different blocking solutions (BSA, serum, commercial blockers) to reduce nonspecific binding.

• Antibody dilution: Perform a dilution series to determine optimal concentration for specific staining.

• Detection system: Compare different detection systems (ABC, polymer-based) for optimal signal-to-noise ratio.

• Counterstains: Select appropriate counterstains that don't interfere with the nuclear localization pattern expected for ERF036.

• Controls: Include positive control tissues known to express ERF036, negative controls without primary antibody, and ideally knockdown/knockout controls.

Studies have shown that antibody performance can vary dramatically between applications, with many antibodies that work well in western blot failing in IHC applications .

What are the best approaches for quantifying ERF036 protein levels in different experimental systems?

Accurate quantification of ERF036 requires careful consideration of several factors:

• Western blot quantification:

  • Use appropriate loading controls (e.g., GAPDH, β-actin)

  • Implement a standard curve of recombinant protein if available

  • Ensure signal is in the linear range of detection

  • Use fluorescent secondary antibodies for wider dynamic range

• Flow cytometry:

  • Standardize using beads with known quantities of fluorochrome

  • Include isotype controls

  • Use median fluorescence intensity rather than mean for more robust measurements

• Mass spectrometry-based quantification:

  • Consider SILAC or TMT labeling for relative quantification

  • Use parallel reaction monitoring (PRM) or selected reaction monitoring (SRM) for targeted quantification

  • Implement heavy-labeled peptide standards for absolute quantification

• Immunohistochemistry quantification:

  • Use digital image analysis software

  • Establish scoring systems (H-score, Allred score)

  • Consider percent positive cells and staining intensity

Each method has strengths and limitations that should be considered in the context of the specific research question.

How can researchers effectively use ERF036 Antibody in combination with other antibodies for multi-parameter analyses?

Multi-parameter analyses require careful planning to avoid technical issues:

• Antibody compatibility considerations:

  • Primary antibody host species should be different to avoid cross-reactivity of secondary antibodies

  • If using primary antibodies from the same species, consider direct conjugation or sequential staining protocols

  • Test for potential cross-reactivity between all antibodies in the panel

• Multiplexed immunofluorescence strategies:

  • Implement spectral unmixing for overlapping fluorophores

  • Consider tyramide signal amplification for sequential staining with same-species antibodies

  • Use nuclear counterstain compatible with nuclear localization of ERF036

• Multi-epitope ligand cartography (MELC):

  • For highly multiplexed analyses, consider photobleaching-based sequential staining

  • Carefully validate signal persistence and potential epitope destruction

• Mass cytometry (CyTOF) adaptation:

  • Metal-conjugated antibodies allow highly multiplexed analyses

  • Requires additional validation of metal-conjugated antibody specificity

Successful multiplexing depends on thorough validation of each antibody individually before combining them, and then additional validation of the combined protocol.

What are the most common causes of false positive and false negative results when using ERF036 Antibody?

Understanding potential sources of error is critical for accurate data interpretation:

Recent studies have demonstrated that even widely used and commercially validated antibodies can produce misleading results, highlighting the importance of implementing multiple controls .

How should researchers interpret ERF036 localization data in stress response experiments?

ERF036 is expected to function as a transcriptional activator responding to stress signals. When interpreting localization data:

• Baseline nuclear localization: Under normal conditions, expect predominant nuclear localization consistent with transcription factor function.

• Stress-induced changes: Monitor potential changes in:

  • Nuclear-cytoplasmic distribution ratio

  • Subnuclear localization patterns (e.g., nucleolar exclusion, chromatin association)

  • Co-localization with other stress response factors

  • Changes in expression level

• Kinetics considerations: Establish a time course to capture transient changes following stress induction.

• Statistical analysis: Quantify localization patterns across multiple cells and biological replicates to ensure reproducibility.

• Controls: Include positive controls (known stress-responsive transcription factors) and negative controls (non-responsive nuclear proteins).

Interpretation should consider that transcription factor dynamics can be rapid and transient, potentially requiring live cell imaging for complete characterization.

What computational tools and databases can help researchers analyze ERF036 binding sites and regulatory networks?

Advanced analysis of ERF036 binding and regulatory networks can leverage these resources:

• ChIP-seq analysis tools:

  • MACS2 for peak calling

  • HOMER for motif analysis

  • deepTools for visualization

  • MEME Suite for motif discovery

• Transcription factor databases:

  • JASPAR for position weight matrices

  • TRANSFAC for binding site information

  • PlantTFDB for plant-specific transcription factor data

• Network analysis tools:

  • Cytoscape for network visualization

  • STRING for protein interaction networks

  • GeneMANIA for functional association networks

• GCC-box motif analysis:

  • Scan for the canonical GCC-box sequence (AGCCGCC)

  • Use position weight matrix models to identify variations

  • Implement tools like FIMO (Find Individual Motif Occurrences) for genome-wide scanning

• Pathway enrichment analysis:

  • GO Term enrichment tools

  • KEGG pathway analysis

  • Reactome for pathway visualization

These computational approaches can help integrate ERF036 binding data with gene expression and other omics datasets to construct comprehensive regulatory networks.

How might ERF036 Antibody be used in studying plant stress responses and climate adaptation?

ERF036 belongs to the ethylene-responsive factor family, which plays crucial roles in plant stress responses. Emerging research applications include:

• Climate change adaptation studies:

  • Analyze ERF036 expression and activity under simulated climate change conditions

  • Compare ERF036 regulation across plant varieties with different stress tolerances

  • Investigate the role of ERF036 in mediating responses to combined stresses (heat + drought)

• Crop improvement applications:

  • Use ERF036 as a molecular marker for stress resistance

  • Screen for natural variants with altered ERF036 activity

  • Monitor effects of genetic modifications on stress response pathways

• Conservation biology:

  • Compare ERF036 regulation across endangered and resilient plant species

  • Investigate evolutionary conservation of stress response mechanisms

• Methodological innovations:

  • Develop ERF036 reporter systems for real-time stress monitoring

  • Implement high-throughput phenotyping with automated immunodetection

  • Create biosensors incorporating anti-ERF036 antibodies for field applications

These applications require rigorous antibody validation to ensure reliable results, particularly when working with diverse plant species where epitope conservation may vary.

What are the current technical limitations in using antibodies like ERF036 for plant research, and how might they be overcome?

Plant-specific antibody research faces several challenges:

• Cell wall barriers:

  • Challenge: Plant cell walls impede antibody penetration

  • Solutions: Optimize cell wall digestion protocols, implement alternative fixation methods, develop cell wall-permeable antibody formats

• Plant-specific autofluorescence:

  • Challenge: Chlorophyll and other pigments create background

  • Solutions: Use spectral unmixing, implement specific blocking steps, select fluorophores with minimal overlap with plant autofluorescence

• Limited commercial antibodies for plant targets:

  • Challenge: Fewer validated antibodies available compared to mammalian research

  • Solutions: Implement rigorous validation protocols, develop community resources for antibody validation data, explore recombinant antibody technologies

• Cross-reactivity with plant-specific compounds:

  • Challenge: Plant secondary metabolites may interfere with antibody binding

  • Solutions: Test multiple extraction methods, implement additional purification steps, include appropriate blocking compounds

• Antibody functionality across species:

  • Challenge: Uncertain epitope conservation across plant species

  • Solutions: Target conserved regions, validate across multiple species, consider synthetic antibody alternatives like aptamers

Emerging technologies like nanobodies, which are smaller and potentially better at penetrating plant tissues, and recombinant antibody libraries specifically designed for plant research, may help address these limitations.

How can advanced imaging techniques enhance ERF036 localization and interaction studies?

Cutting-edge imaging approaches can provide deeper insights into ERF036 biology:

• Super-resolution microscopy applications:

  • Stimulated emission depletion (STED) microscopy to resolve subnuclear localization

  • Single-molecule localization microscopy (PALM/STORM) to track individual ERF036 molecules

  • Structured illumination microscopy (SIM) for improved resolution of nuclear distribution patterns

• Live cell imaging strategies:

  • Fluorescent protein fusions to track dynamics in real-time

  • Optogenetic approaches to manipulate ERF036 activity with light

  • FRAP (Fluorescence Recovery After Photobleaching) to measure mobility and binding kinetics

• Proximity labeling approaches:

  • BioID or TurboID fusions to identify proteins in close proximity to ERF036

  • APEX2-based techniques for spatially restricted protein labeling

  • Integration with mass spectrometry for comprehensive interaction profiling

• Correlative light and electron microscopy (CLEM):

  • Combine fluorescence localization with ultrastructural context

  • Immunogold labeling for high-resolution localization

  • Cryo-electron tomography for 3D structural context

• Volumetric imaging:

  • Light sheet microscopy for whole-tissue analysis with minimal photodamage

  • Tissue clearing techniques compatible with antibody labeling

  • 3D reconstruction of ERF036 distribution in intact plant tissues

These advanced imaging approaches can reveal novel aspects of ERF036 biology but require careful optimization and validation of the antibody under each specific imaging condition.