ERF013 Antibody

What is ERF013 and why is it significant in plant biology research?

ERF013 is an ethylene response factor transcription factor in Arabidopsis thaliana that serves as a critical regulatory node in multiple plant processes. Research has shown that ERF013 negatively regulates defense against the bacterial pathogen Pseudomonas syringae and functions at the junction of signaling pathways for multiple plant hormones, including salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and abscisic acid (ABA) . More recent studies have revealed that ERF013 also plays a crucial role in lateral root development by preventing lateral root emergence until it is degraded through auxin-activated signaling pathways . The multifunctional nature of ERF013 makes it an important target for understanding how plants coordinate responses to both biotic and abiotic stresses.

What types of ERF013 antibodies are currently available for research?

Currently, the primary type of ERF013 antibody available for research is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana ERF013 protein. These antibodies are typically purified using antigen affinity methods and supplied in a storage buffer containing preservatives (e.g., 0.03% Proclin 300), 50% glycerol, and 0.01M PBS at pH 7.4 . Commercial ERF013 antibodies are generally validated for applications such as ELISA and Western blotting. At present, there do not appear to be commercially available monoclonal antibodies against ERF013, which could offer greater specificity for certain specialized applications. Researchers should verify the validation status of any antibody for their specific application before proceeding with experiments.

What are the standard applications of ERF013 antibody in plant research?

ERF013 antibodies can be utilized in several key applications in plant molecular biology research:

• Western Blotting (WB): For detecting and quantifying ERF013 protein levels in plant tissue extracts, particularly useful for studying expression changes under different conditions or treatments .

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of ERF013 in plant samples with potentially higher sensitivity than Western blotting .

• Immunoprecipitation (IP): For isolating ERF013 and its interacting protein partners, as demonstrated in studies that identified interactions between ERF013 and E3 ubiquitin ligases MAC3A and MAC3B .

• Chromatin Immunoprecipitation (ChIP): For identifying DNA sequences bound by ERF013, helping to elucidate its role as a transcription factor in regulating target genes.

• Immunohistochemistry/Immunofluorescence: For visualizing the tissue and subcellular localization of ERF013 protein, though this application may require additional optimization.

How should Western blotting protocols be optimized for ERF013 detection?

Optimizing Western blotting for ERF013 detection requires careful consideration of several parameters:

Sample Preparation:

• Extract proteins from plant tissues using a buffer containing protease inhibitors and phosphatase inhibitors (especially important since ERF013 can be phosphorylated by MPK14) .

• Include reducing agents like DTT or β-mercaptoethanol to ensure proper protein denaturation.

• Consider using specialized extraction protocols for nuclear proteins, as ERF013 is a transcription factor.

Gel and Transfer Parameters:

• Use 10-12% SDS-PAGE gels for optimal resolution of ERF013.

• PVDF membranes may provide better protein retention than nitrocellulose.

• Optimize transfer conditions (voltage, time, buffer composition) based on your equipment.

Antibody Conditions:

• Begin with manufacturer's recommended dilution (typically 1:1000) and optimize as needed.

• Test both overnight incubation at 4°C and shorter incubations at room temperature.

• Compare blocking reagents (5% non-fat milk vs. 3-5% BSA) to determine which gives lowest background.

Controls:

• Include positive control (tissue known to express ERF013)

• Include negative control (tissue where ERF013 is absent or knocked down)

• Consider including recombinant ERF013 protein as a size reference.

What approaches can validate the specificity of ERF013 antibody?

Validating antibody specificity is crucial for reliable research results. For ERF013 antibody, consider these approaches:

Genetic Controls:

• Test the antibody on tissues from ERF013 knockout/knockdown plants (e.g., T-DNA insertion lines).

• The antibody should show significantly reduced or absent signal in these plants.

Overexpression Controls:

• Test on tissues overexpressing ERF013 (e.g., 35S:ERF013), which should show enhanced signal.

Peptide Competition Assay:

• Pre-incubate the antibody with excess immunizing peptide/protein.

• This should block specific binding and eliminate the true signal while non-specific binding would remain.

Western Blot Analysis:

• Verify that the antibody detects a band of the expected molecular weight.

• Multiple or unexpected bands may indicate cross-reactivity with other proteins.

Immunoprecipitation-Mass Spectrometry:

• Perform IP with the ERF013 antibody followed by mass spectrometry.

• This can confirm that the antibody is primarily pulling down ERF013 rather than other proteins.

Cross-Reactivity Testing:

• Test the antibody against related Arabidopsis ERF family members expressed heterologously.

• This establishes whether the antibody cross-reacts with other ERF proteins.

What are the optimal storage and handling conditions for ERF013 antibody?

Proper storage and handling of ERF013 antibody is essential for maintaining its activity:

Storage Conditions:

• Store at -20°C or -80°C upon receipt, avoiding repeated freeze-thaw cycles .

• Aliquot the antibody into smaller volumes before freezing to minimize freeze-thaw cycles.

• Keep a working aliquot at 4°C for up to two weeks if used regularly.

Working Solutions:

• Prepare working dilutions fresh on the day of use rather than storing diluted antibody.

• Use high-quality, sterile buffers for antibody dilution.

• When diluting, use the same buffer composition recommended by the manufacturer.

Handling Protocols:

• Thaw frozen antibody on ice and centrifuge briefly before opening.

• Use sterile technique to prevent microbial contamination.

• Avoid vigorous vortexing which can damage antibody structure; instead, mix by gentle inversion or flicking.

Quality Control:

• Maintain a record of antibody performance over time to detect any degradation.

• Include a known positive control in each experiment to verify antibody activity.

• Consider testing new lots against previous lots before discarding older material.

How can ERF013 antibody be used to study protein-protein interactions?

ERF013 antibody enables several sophisticated approaches for investigating protein-protein interactions:

Co-Immunoprecipitation (Co-IP):

• Use ERF013 antibody to pull down ERF013 and analyze co-precipitated proteins.

• This approach successfully identified interactions between ERF013 and E3 ubiquitin ligases MAC3A and MAC3B .

• Protocol steps include:
  a. Prepare plant tissue lysate in a non-denaturing buffer
  b. Pre-clear with protein A/G beads
  c. Incubate with ERF013 antibody
  d. Capture antibody-protein complexes with protein A/G beads
  e. Wash thoroughly to remove non-specific interactions
  f. Elute and analyze by Western blot or mass spectrometry

Immunoprecipitation-Mass Spectrometry (IP-MS):

• More comprehensive approach for identifying multiple interaction partners.

• Research has used this technique to discover ERF013's interaction with MAC3A and MAC3B .

• Consider crosslinking proteins before lysis to capture transient interactions.

Proximity Ligation Assay (PLA):

• For visualizing protein interactions in situ within plant tissues or cells.

• Requires both ERF013 antibody and antibody against the suspected interaction partner.

• Can confirm interactions identified by Co-IP in their native cellular context.

Sequential Co-IP:

• For studying complex protein assemblies involving ERF013.

• First IP with ERF013 antibody, followed by a second IP with antibody against another suspected complex component.

Table 1: Comparison of Techniques for Studying ERF013 Protein Interactions

How can ERF013 antibody be used to study its role in plant immunity?

ERF013 negatively regulates defense against Pseudomonas syringae through hormone signaling crosstalk . To investigate its role in immunity:

Expression Analysis During Infection:

• Monitor ERF013 protein levels during pathogen infection using Western blotting.

• Compare expression in compatible versus incompatible plant-pathogen interactions.

• Collect tissue at multiple time points post-infection to track dynamic changes.

Subcellular Localization Changes:

• Use immunofluorescence with ERF013 antibody to track its localization before and after pathogen challenge.

• Co-stain with markers for different cellular compartments to detect translocation events.

Chromatin Immunoprecipitation (ChIP):

• Identify ERF013 target genes during immune responses with this protocol:
  a. Crosslink protein-DNA interactions in plant tissue
  b. Fragment chromatin
  c. Immunoprecipitate with ERF013 antibody
  d. Purify and analyze bound DNA by qPCR or sequencing

• This can reveal how pathogen challenge alters ERF013's interaction with target promoters.

Protein Complex Analysis:

• Immunoprecipitate ERF013 before and after pathogen treatment.

• Identify differentially associated proteins by mass spectrometry.

• This can reveal how the ERF013 interaction network changes during immune responses.

Hormone Response Studies:

• Treat plants with different hormones (SA, JA, ET, ABA) and monitor ERF013 protein levels and modifications.

• This helps understand how hormone crosstalk regulates ERF013 function in immunity.

What approaches can be used to study ERF013 phosphorylation?

ERF013 is phosphorylated by MPK14 kinase, which affects its stability and function . Several approaches can be used to study this phosphorylation:

Phos-tag SDS-PAGE:

• Use regular ERF013 antibody with Phos-tag acrylamide gels.

• Phosphorylated forms migrate more slowly, creating distinct bands.

• Compare migration patterns with and without phosphatase treatment.

Immunoprecipitation followed by Phospho-staining:

In vitro Kinase Assays:

• Immunoprecipitate ERF013 from plant tissues.

• Incubate with purified MPK14 and ATP.

• Detect phosphorylation using phospho-specific antibodies or radioactive ATP.

Mass Spectrometry Approach:

• Immunoprecipitate ERF013 from plant tissues under different conditions.

• Perform mass spectrometry to identify phosphorylation sites.

• Compare phosphorylation patterns after various treatments (e.g., auxin, pathogen exposure).

Pharmacological Studies:

• Treat plants with kinase inhibitors or phosphatase inhibitors.

• Immunoprecipitate ERF013 and analyze phosphorylation status.

• This approach can help determine the dynamics and regulation of ERF013 phosphorylation.

What are common causes of inconsistent ERF013 antibody staining patterns?

Several factors can contribute to inconsistent ERF013 antibody staining:

Biological Variability:

• ERF013 expression is regulated by hormones and stress conditions .

• Plants at different developmental stages may have varying ERF013 levels.

• Solution: Standardize plant growth conditions and developmental stages across experiments.

Protein Degradation:

• ERF013 is targeted for degradation by MAC3A and MAC3B ubiquitin ligases .

• Different tissues might have different rates of ERF013 turnover.

• Solution: Include proteasome inhibitors (e.g., MG132) in extraction buffers.

Post-translational Modifications:

• Phosphorylation of ERF013 by MPK14 may affect antibody recognition.

• Other uncharacterized modifications might also influence antibody binding.

• Solution: Consider the impact of treatments on post-translational modifications and test multiple extraction conditions.

Technical Variables:

• Inconsistent blocking or washing steps.

• Variable incubation times or temperatures.

• Solution: Standardize protocols and use automated systems where possible.

Antibody Quality:

• Batch-to-batch variations in polyclonal antibodies.

• Antibody degradation due to improper storage.

• Solution: Validate each new antibody lot; store according to manufacturer recommendations.

Table 2: Troubleshooting ERF013 Antibody Issues

How can ERF013 expression levels be quantified accurately?

Accurate quantification of ERF013 protein levels requires careful experimental design and analysis:

Western Blot Quantification:

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

• Consider using stain-free technology for total protein normalization as an alternative to single protein loading controls.

• Ensure signal is in the linear range of detection by performing a dilution series.

• Use digital imaging and software analysis (e.g., ImageJ) for densitometry.

• Always include a standard curve using recombinant protein for absolute quantification.

ELISA-based Quantification:

• Develop a sandwich ELISA if two antibodies recognizing different ERF013 epitopes are available.

• Include a standard curve using recombinant ERF013 protein.

• Process all samples identically to minimize technical variation.

Statistical Considerations:

• Include at least three biological replicates in all experiments.

• Perform technical replicates for each biological sample.

• Apply appropriate statistical analyses (e.g., ANOVA, t-tests) based on experimental design.

• Report not just p-values but also effect sizes and confidence intervals.

Normalization Strategies:

• Normalize to total protein amount loaded rather than single reference proteins when possible.

• For tissue sections, normalize to area or specific cell types.

• Consider the impact of treatments on reference proteins before selecting normalization method.

Complementary Approaches:

• Correlate protein levels with mRNA expression to identify post-transcriptional regulation.

• Consider absolute quantification methods like selected reaction monitoring (SRM) mass spectrometry for highest accuracy.

What controls should be included when using ERF013 antibody?

Proper controls are essential for reliable results with ERF013 antibody:

Positive Controls:

• Samples known to express ERF013 (e.g., specific tissues based on literature).

• Recombinant ERF013 protein (if available).

• Transgenic plants overexpressing ERF013.

Negative Controls:

• ERF013 knockout or knockdown plant tissues.

• Tissues known not to express ERF013 based on previous studies.

• Primary antibody omission control to detect non-specific binding of secondary antibody.

Specificity Controls:

• Peptide competition/blocking with immunizing antigen.

• Isotype control (irrelevant antibody of same isotype and concentration).

• Testing on multiple Arabidopsis accessions for consistent results.

Technical Controls:

• Loading controls for Western blots (e.g., actin, tubulin, or total protein stain).

• Staining controls for immunohistochemistry (e.g., nuclear stain).

• Unstained samples to assess autofluorescence in fluorescence applications.

Application-Specific Controls:

• For ChIP: Input DNA, IgG control IP, no-antibody control.

• For Co-IP: IgG control, bead-only control, reverse IP with antibody against interaction partner.

• For immunofluorescence: Secondary antibody only control, autofluorescence controls.

How can ERF013 antibody contribute to understanding plant stress responses?

ERF013 antibody can enable several novel research directions for understanding plant stress responses:

Temporal-Spatial Dynamics:

• Track ERF013 protein localization and abundance during different stress conditions.

• Correlate with physiological responses to identify critical time points for stress response regulation.

• The antibody allows visualization of protein movement that cannot be achieved with transcript analysis alone.

Multi-Stress Integration:

• ERF013 functions at the intersection of multiple hormone pathways .

• Use the antibody to study how ERF013 integrates signals from simultaneous biotic and abiotic stresses.

• This could reveal prioritization mechanisms in plant stress responses.

Protein Modification Landscapes:

• Beyond phosphorylation by MPK14 , ERF013 may undergo other modifications.

• Combine ERF013 immunoprecipitation with mass spectrometry to identify stress-induced modifications.

• This could reveal previously unknown regulatory mechanisms affecting ERF013 function.

Protein Complex Dynamics:

• Different stresses may induce formation of different ERF013-containing protein complexes.

• Use the antibody for comparative proteomics across stress conditions.

• This approach can identify stress-specific co-factors that modulate ERF013 function.

Chromatin Association Patterns:

• ChIP-seq with ERF013 antibody under different stress conditions.

• May reveal stress-specific target genes and binding patterns.

• Could identify novel stress response pathways regulated by ERF013.

What emerging techniques could enhance ERF013 antibody-based research?

Several cutting-edge techniques could significantly advance ERF013 research when combined with antibody-based approaches:

Single-Cell Protein Analysis:

• Adaptation of single-cell Western blotting for plant cells.

• Mass cytometry (CyTOF) with metal-conjugated ERF013 antibodies.

• Could reveal cell-type-specific regulation of ERF013 within complex tissues.

Spatial Transcriptomics Integration:

• Combine immunohistochemistry using ERF013 antibody with spatial transcriptomics.

• Correlate protein localization with transcriptional landscapes.

• May identify spatial domains of ERF013 activity within plant organs and tissues.

CRISPR-Based Tagging with Antibody Validation:

• Use CRISPR to add small epitope tags to endogenous ERF013.

• Validate with both tag antibodies and ERF013 antibody.

• Allows live tracking while confirming results with native protein detection.

Proximity Labeling Technologies:

• BioID or APEX2 fused to ERF013 to identify proximal proteins in vivo.

• Use ERF013 antibody to validate identified interactions.

• Can capture transient interactions missed by traditional co-immunoprecipitation.

Cryo-Electron Microscopy:

• Immunoprecipitate ERF013 complexes and analyze by cryo-EM.

• Could provide structural insights into ERF013 interactions with DNA and protein partners.

• May reveal conformational changes associated with post-translational modifications.

Table 3: Emerging Technologies for ERF013 Research