ERF015 Antibody

What is ERF015 and what role does it play in plant biology?

ERF015 (Ethylene-responsive transcription factor ERF015) is a member of the AP2/ERF superfamily of transcription factors in plants. These transcription factors regulate gene expression in response to ethylene and various environmental stresses. In Arabidopsis thaliana, ERF015 (At4g31060) functions as a key regulator in plant growth, development, and stress responses .
The homolog in Marchantia polymorpha (MpERF15) has been specifically identified as essential for gemmaling regeneration following tissue damage. Research has demonstrated that MpERF15 is activated immediately after wounding and drives regeneration by activating an oxylipin biosynthesis feedback loop .

What applications is the ERF015 antibody suitable for?

The ERF015 antibody is primarily used in the following experimental applications:

How specific is the ERF015 antibody and what controls should I use?

Specificity is crucial for antibody-based experiments. For ERF015 antibody, it's important to note:

• The antibody specifically detects ethylene-responsive transcription factor ERF015 in Arabidopsis thaliana and potentially in other closely related plant species.

• Recommended controls include:

  • Pre-immune serum (negative control)

  • Recombinant ERF015 protein (positive control)

  • ERF015 knockout/knockdown plant tissues (negative control)

  • Closely related ERF transcription factors to test for cross-reactivity
    Based on practices with similar antibodies like IRF5 antibodies , it's advisable to run parallel tests with knockout/knockdown samples to verify specificity, as antibody specificity can vary significantly even among products targeting the same protein.

How does ERF015 function differ from other ERF family members in plants?

ERF015 has distinct functions compared to other ERF family members:

• While many ERFs like ERF1-2 and ERF053 are involved in stress responses, ERF015 appears to have a more specialized role in development and regeneration processes .

• Research on the Marchantia homolog (MpERF15) shows it functions in a feedback loop with oxylipins. When MpERF15 is overexpressed:

  • It results in approximately eight apical notches in 10-day-old thallus, compared to four in wild-type

  • It induces morphological changes including dwarfed compact thallus structure

  • It increases the number of meristematic notches and EdU-positive concave structures

  • These phenotypes are dependent on MpCOI1, as MpCOI1 knockout rescues the phenotype

• Unlike Arabidopsis ERF109, MpERF15 activates an oxylipin biosynthesis feedback loop, where:

  • MpERF15 enhances biosynthesis of dinor-OPDA (dn-OPDA)

  • Overexpression of MpERF15 stimulates endogenous MpERF15 transcription in a MpCOI1-dependent manner
    This distinct regulatory mechanism differentiates ERF015 from other ERF family members and highlights its specialized role in plant regeneration processes.

What are the challenges in designing specific antibodies against ERF transcription factors?

Designing specific antibodies against ERF transcription factors presents several challenges:

• High sequence homology: ERF family members share conserved DNA-binding domains, making it difficult to generate antibodies that don't cross-react with related proteins. This is similar to challenges faced with other transcription factor families like IRF, where research has shown many commercially available antibodies lack specificity .

• Structural similarity: The ETS domain shared among ERF family members recognizes similar DNA sequences (GGAA/T), creating structural similarities that can lead to cross-reactivity .

• Low expression levels: Transcription factors are often expressed at low levels, requiring highly sensitive antibodies.

• Solution approach: Leveraging modern antibody engineering techniques, such as:

  • Recombinant antibody technology for better reproducibility

  • Phage display methods to select antibodies with higher specificity

  • Affinity maturation to enhance binding properties

  • Using AI-driven approaches such as RFdiffusion to design antibodies with specific binding properties
    Researchers should consider these challenges when selecting or designing antibodies against ERF015 and validate specificity through appropriate controls.

How can I use ERF015 antibody to study the wound healing and regeneration pathway in plants?

To study wound healing and regeneration pathways using ERF015 antibody, a comprehensive experimental approach is recommended:

• Temporal expression analysis:

  • Perform western blotting with ERF015 antibody on tissue samples collected at different time points after wounding (e.g., 0, 1, 3, 6, 12, 24, 48 hours)

  • Quantify protein levels using densitometry and normalize to housekeeping proteins

• Spatial expression analysis:

  • Use immunohistochemistry or immunofluorescence to visualize ERF015 localization in wounded tissues

  • Compare expression patterns between wounded and unwounded tissues

  • For Marchantia studies, focus on meristematic regions where EdU staining has shown cell cycle activity

• Functional analysis:

  • Create ERF015 knockout/knockdown lines (validate using the antibody)

  • Quantify regeneration defects (as seen with Mp ko erf15 mutants)

  • Perform complementation studies by reintroducing ERF015

• Pathway interaction studies:

  • Co-immunoprecipitation with ERF015 antibody to identify interacting proteins

  • ChIP-seq to identify ERF015 binding sites in the genome

  • Combine with transcriptomics to identify downstream targets

• Feedback loop verification:

  • Monitor ERF015 expression in response to exogenous application of pathway components (e.g., OPDA/dn-OPDA for Marchantia)

  • Validate the COI1-dependent feedback loop as demonstrated in Marchantia
    Research on MpERF15 has shown that it's instantly activated after wounding and is essential for regeneration, making it an excellent target for studying these processes .

What are the optimal conditions for using ERF015 antibody in Western blot analysis?

For optimal Western blot results with ERF015 antibody, follow these methodological guidelines:

• Sample preparation:

  • Extract total protein from plant tissues using a buffer containing:

    • 50 mM Tris-HCl (pH 7.5)

    • 150 mM NaCl

    • 1% Triton X-100

    • 0.1% SDS

    • Protease inhibitor cocktail

  • Quantify protein concentration (Bradford or BCA assay)

  • Use 20-50 μg total protein per lane

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gel

  • Transfer to PVDF membrane (preferable over nitrocellulose for transcription factors)

  • Verify transfer efficiency with Ponceau S staining

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Incubate with ERF015 antibody at 1:1000 dilution in blocking buffer overnight at 4°C

  • Wash 3-5 times with TBST

  • Incubate with HRP-conjugated secondary antibody at 1:5000 for 1 hour

  • Wash thoroughly (5-6 times with TBST)

• Detection and troubleshooting:

  • Use ECL substrate for detection

  • Include molecular weight markers

  • Expected molecular weight for ERF015: approximately 30-40 kDa

  • If non-specific bands appear, try higher antibody dilution (1:2000) or more stringent washing
    Based on experiences with similar transcription factor antibodies, titration of antibody concentration is often necessary for optimal signal-to-noise ratio .

How can I validate the specificity of ERF015 antibody for my experimental system?

Rigorous validation of antibody specificity is essential for reliable research outcomes. For ERF015 antibody, implement the following validation strategy:

How can I optimize the ERF015 antibody for chromatin immunoprecipitation (ChIP) experiments?

Optimizing ERF015 antibody for ChIP requires careful method development:

• Antibody suitability assessment:

  • Not all antibodies work well for ChIP, even if they perform in Western blot

  • Verify the antibody can recognize native (non-denatured) protein

  • Test immunoprecipitation (IP) capability before proceeding to ChIP

• Crosslinking optimization:

  • For plant tissues, start with 1% formaldehyde for 10 minutes at room temperature

  • Test different crosslinking times (5-15 minutes) to optimize chromatin preparation

  • Quench with 0.125 M glycine for 5 minutes

• Chromatin preparation:

  • Sonicate to generate DNA fragments of 200-500 bp

  • Verify fragment size on agarose gel

  • Pre-clear chromatin with protein A/G beads to reduce background

• IP conditions optimization:

  • Test different antibody amounts (2-10 μg per IP reaction)

  • Optimize incubation time (overnight at 4°C is standard)

  • Include appropriate controls:

    • Input DNA (non-immunoprecipitated)

    • IgG control (non-specific antibody)

    • IP in knockout/knockdown tissues

• Washing and elution:

  • Use increasingly stringent wash buffers

  • Monitor background with qPCR of negative control regions

• Target validation:

  • Design primers for known ERF binding sites containing the GCC-box motif

  • Use qPCR to verify enrichment at these sites

  • Validate findings by comparing enrichment patterns between wild-type and erf015 mutant plants
    Based on experience with transcription factor ChIP protocols like those for EBF1 , optimizing antibody concentration and chromatin preparation are the most critical steps for successful ChIP experiments.

What approaches can be used to improve ERF015 antibody specificity if cross-reactivity is observed?

If cross-reactivity is observed with ERF015 antibody, several approaches can be implemented to improve specificity:

• Antibody purification techniques:

  • Affinity purification against the immunizing peptide/protein

  • Negative selection against cross-reactive proteins

  • These methods can significantly enhance specificity, as demonstrated with IRF5 antibodies

• Epitope-focused approach:

  • Design new antibodies against unique regions of ERF015

  • Avoid conserved domains shared with other ERF family members

  • Target N- or C-terminal regions which typically show greater sequence divergence

• Modern antibody engineering:

  • Apply phage display selection under more stringent conditions to isolate more specific binders

  • Implement affinity maturation to improve binding characteristics while maintaining specificity

  • This approach has proven successful in generating highly specific antibodies against similar targets

• Alternative detection strategies:

  • Use epitope tagging in transgenic plants (e.g., HA, FLAG, GFP tags)

  • Employ CRISPR/Cas9 to add endogenous tags to ERF015

  • These approaches circumvent specificity issues by using well-characterized tag antibodies

• AI-assisted antibody design:

  • Utilize computational tools like RFdiffusion to design antibodies with customized specificity profiles

  • These methods can create antibodies with high affinity for particular target ligands while excluding others
    Research has shown that careful antibody selection or redesign can significantly improve experimental outcomes, particularly for transcription factors with many family members .

How should I design experiments to study ERF015's role in stress response pathways?

A comprehensive experimental design to study ERF015's role in stress responses should include:

• Expression analysis under various stresses:

  • Subject plants to different stresses (drought, salinity, heat, cold, pathogen infection)

  • Monitor ERF015 protein levels by Western blot at multiple time points

  • Compare with mRNA expression by qRT-PCR to understand transcriptional vs. post-transcriptional regulation

• Genetic manipulation approach:

  • Generate and characterize ERF015 knockout, knockdown, and overexpression lines

  • Phenotype these lines under normal and stress conditions

  • Key parameters to measure:

    • Growth parameters (fresh weight, dry weight, root length)

    • Physiological responses (relative water content, electrolyte leakage, photosynthetic efficiency)

    • Stress marker genes expression

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation with ERF015 antibody followed by mass spectrometry

  • Validate key interactions by yeast two-hybrid or bimolecular fluorescence complementation

  • Focus on interactions that change under stress conditions

• Genome-wide binding site analysis:

  • Conduct ChIP-seq using ERF015 antibody under normal and stress conditions

  • Identify stress-specific binding patterns

  • Correlate with transcriptomic changes using RNA-seq

• Pathway integration:

  • Use pharmacological treatments to activate/inhibit related signaling pathways

  • Monitor ERF015 protein levels, localization, and post-translational modifications

  • This approach can help position ERF015 within the broader stress signaling network
    Based on research with MpERF15, consider investigating potential feedback loops involving phytohormones or secondary messengers, as these have been shown to be important for ERF function .

What are common pitfalls when working with ERF015 antibody and how can they be avoided?

Researchers may encounter several challenges when working with ERF015 antibody. Here are common pitfalls and solutions:

• Non-specific binding:

  • Pitfall: Multiple bands in Western blot or high background in immunostaining

  • Solution: Optimize antibody concentration, increase blocking time/concentration, and use more stringent washing

  • Consider additional validation with knockout/knockdown samples as recommended for IRF5 antibodies

• Epitope masking:

  • Pitfall: Reduced or absent signal due to protein-protein interactions or post-translational modifications

  • Solution: Vary extraction conditions (different detergents, salt concentrations) and test denaturing vs. native conditions

  • For fixed tissues, optimize antigen retrieval methods

• Batch-to-batch variation:

  • Pitfall: Inconsistent results between antibody lots

  • Solution: Reserve single lots for entire project series, validate each new lot against previous ones

  • Consider recombinant antibodies which typically show better reproducibility

• Low signal issues:

  • Pitfall: Weak or undetectable signal in experiments

  • Solution: Increase protein loading for Western blots, optimize extraction to preserve transcription factors

  • Use signal enhancement systems (amplification kits, more sensitive detection substrates)

• Antibody degradation:

  • Pitfall: Declining performance over time

  • Solution: Aliquot antibody upon receipt, avoid freeze-thaw cycles

  • Store according to manufacturer's recommendations (typically -20°C or -80°C)

• Cross-reactivity with related proteins:

  • Pitfall: Inability to distinguish between ERF family members

  • Solution: Use complementary approaches (gene expression, tagged protein expression)

  • When possible, design experiments with genetic validation to confirm specificity
    Lessons from antibody validation studies emphasize the importance of including proper controls in every experiment and maintaining detailed records of antibody performance across different experimental conditions .

How can I design a multiplexed immunoassay to simultaneously detect ERF015 and other stress-related transcription factors?

Designing a multiplexed immunoassay for ERF015 and other transcription factors requires careful planning:

• Antibody selection criteria:

  • Choose antibodies raised in different host species to avoid cross-reactivity of secondary antibodies

  • Verify that all selected antibodies work under similar conditions

  • Test each antibody individually before combining them

• Immunofluorescence multiplex approach:

  • Use fluorophore-conjugated secondary antibodies with non-overlapping emission spectra

  • Apply sequential staining protocol to minimize cross-reactivity:

    1. Incubate with first primary antibody

    2. Wash thoroughly

    3. Incubate with corresponding fluorophore-conjugated secondary antibody

    4. Wash thoroughly

    5. Repeat for additional antibodies

• Western blot multiplexing strategies:

  • Method 1: Strip and reprobe membrane

    • Verify stripping efficiency between antibody applications

    • Start with antibodies requiring highest sensitivity

  • Method 2: Cut membrane based on molecular weight

    • Requires proteins of interest to have sufficiently different sizes

  • Method 3: Use fluorescent secondary antibodies on single membrane

    • Similar to immunofluorescence approach but applied to Western blots

• Flow cytometry multiplexing:

  • For single-cell suspensions from plant protoplasts

  • Use directly conjugated primary antibodies when available

  • Employ careful compensation controls to account for spectral overlap

• Data analysis considerations:

  • Include single-target controls to establish baseline signals

  • Apply appropriate normalization methods for cross-experiment comparisons

  • Use correlation analysis to identify co-regulated factors

• Validation strategy:

  • Confirm multiplex results with single-plex assays

  • Use genetic knockouts to verify specificity of each signal in the multiplex assay
    Similar approaches have been successfully implemented for analyzing multiple transcription factors in various biological systems, including studies on EBF1 and related factors .

How can AI-driven approaches improve the design and application of antibodies against ERF015?

AI-driven approaches offer significant advantages for antibody design and application:

• Structure-informed antibody design:

  • AI models like RFdiffusion can generate antibody blueprints that bind user-specified targets

  • These models can design antibody loops—the intricate, flexible regions responsible for binding

  • For ERF015, this could enable creation of antibodies that specifically distinguish it from closely related ERF proteins

• Target-specific optimization:

  • Computational models can identify optimal epitopes unique to ERF015

  • Machine learning algorithms can predict antibody performance across applications (Western blot, IHC, ChIP)

  • This approach has been successfully applied to design antibodies against influenza hemagglutinin and bacterial toxins

• Affinity maturation in silico:

  • AI systems can model antibody-antigen interactions and suggest mutations to improve binding

  • This eliminates the need for labor-intensive experimental affinity maturation

  • Research shows that computational design of antibodies with customized specificity profiles is feasible

• Cross-reactivity prediction and elimination:

  • AI can scan proteomes to identify potential cross-reactive proteins

  • Antibody designs can be refined to minimize unwanted interactions

  • This approach is particularly valuable for ERF family members which share conserved domains

• Application-specific optimization:

  • AI can design antibodies optimized for specific applications (e.g., ChIP-seq vs. Western blot)

  • Models can incorporate parameters such as epitope accessibility in different experimental conditions

• Future implementation:

  • Integration with laboratory automation for rapid design-test cycles

  • Combination with high-throughput screening to validate AI predictions

  • Development of custom antibodies against post-translationally modified ERF015
    Recent advances demonstrate that AI-designed antibodies can achieve binding affinities comparable to or better than traditional methods, suggesting significant potential for improving ERF015 antibody specificity and performance .

What emerging technologies could enhance the detection sensitivity and specificity for ERF015 in plant tissues?

Several emerging technologies show promise for enhancing ERF015 detection:

• Proximity ligation assay (PLA):

  • Enables visualization of protein-protein interactions in situ

  • Provides single-molecule sensitivity through rolling circle amplification

  • Can detect ERF015 interactions with DNA or protein partners in fixed plant tissues

  • Requires two antibodies binding neighboring epitopes, reducing false positives

• Mass cytometry (CyTOF):

  • Uses metal-tagged antibodies instead of fluorophores

  • Eliminates spectral overlap issues in multiplexed detection

  • Allows simultaneous detection of >40 proteins

  • Could be adapted for plant single-cell suspensions to profile ERF015 alongside numerous signaling components

• Super-resolution microscopy techniques:

  • STORM, PALM, or STED microscopy surpass diffraction limits

  • Enable visualization of ERF015 localization with nanometer precision

  • Can reveal subnuclear distribution patterns not visible with conventional microscopy

  • Particularly valuable for studying transcription factor clustering at enhancer regions

• Nanobody technology:

  • Single-domain antibodies derived from camelid antibodies

  • Smaller size (15 kDa vs. 150 kDa) enables better tissue penetration

  • Can access epitopes not available to conventional antibodies

  • May improve nuclear localization for transcription factor detection

• CRISPR-based tagging:

  • Endogenous tagging of ERF015 with split fluorescent proteins or epitope tags

  • Preserves natural expression levels and regulation

  • Eliminates antibody specificity concerns

  • Can be combined with tissue-specific promoters for cell-type-specific studies

• Digital protein profiling:

  • Single-molecule arrays (Simoa) for ultrasensitive protein detection

  • Can detect proteins at femtomolar concentrations

  • Potential adaptation for plant tissue extracts could enable detection of very low-abundance transcription factors
    These technologies represent significant advancements over traditional immunodetection methods and could revolutionize our ability to study low-abundance transcription factors like ERF015 in complex plant tissues.

How can ERF015 antibody be used in comparative studies across different plant species?

Using ERF015 antibody for cross-species studies requires careful consideration:

• Sequence conservation analysis:

  • Perform multiple sequence alignment of ERF015 homologs across target species

  • Identify the antibody epitope region and assess conservation

  • Predict cross-reactivity based on sequence similarity

  • Example: If studying ERF015 across Brassicaceae family members, alignment would reveal high conservation (~80-90%) suggesting potential cross-reactivity

• Validation strategy for cross-species applications:

  • Test antibody on recombinant proteins from each species if available

  • Perform Western blot on tissue extracts from different species

  • Include positive controls (Arabidopsis) and negative controls (knockout lines if available)

  • Verify specificity using orthogonal methods (mRNA expression, tagged proteins)

• Experimental design for comparative studies:

  • Standardize tissue collection, storage, and processing across species

  • Normalize protein loading carefully (using conserved housekeeping proteins)

  • Consider developmental equivalence rather than chronological age when comparing species

  • Document tissue fixation differences that might affect epitope accessibility

• Data interpretation considerations:

  • Account for potential differences in antibody affinity across species

  • Consider evolutionary divergence in protein function/regulation

  • Interpret negative results cautiously (absence of signal could reflect epitope divergence rather than protein absence)

• Integrative approach:

  • Combine antibody-based detection with transcriptomic data

  • When possible, complement with species-specific genetic approaches

  • Create phylogenetic trees of expression patterns and protein function

• Application example:

  • Study of ERF015's role in regeneration could compare Arabidopsis thaliana and Marchantia polymorpha

  • MpERF15 has been shown to be critical for regeneration through an oxylipin biosynthesis feedback loop

  • Comparative analysis could reveal conservation or divergence of this pathway across plant lineages This approach has been successfully applied in studies comparing transcription factor function across species, providing insights into the evolution of regulatory networks in plants.