ERF060 Antibody

What is ERF060 and why is it important in plant research?

ERF060 (also identified as O65665) is an antibody that targets the Ethylene Response Factor 6 (ERF6) in Arabidopsis thaliana. This transcription factor is critical in plant stress responses, particularly in pathogen defense and environmental adaptation . ERF6 functions as a substrate of MPK3 and MPK6, playing important roles downstream of the MPK3/MPK6 cascade in regulating plant defense against fungal pathogens . Research has demonstrated that phosphorylation of ERF6 by MPK3/MPK6 increases its protein stability in vivo, making it a valuable target for studying signal transduction in plant defense mechanisms.

What are the primary applications of ERF060 antibody in plant molecular biology?

ERF060 antibody is primarily used to:

• Detect and quantify ERF6 protein expression in plant tissues

• Monitor phosphorylation status of ERF6 in response to stressors

• Study protein-protein interactions involving ERF6

• Investigate retrograde signaling pathways from chloroplast to nucleus

• Examine plant defense activation following pathogen exposure
  The antibody has proven particularly valuable in studying the rapid transcriptional responses to environmental stimuli, as ERF6 shows significant upregulation within 10 minutes of high light exposure and during early stages of pathogen infection .

How should I design experiments to detect ERF6 phosphorylation using ERF060 antibody?

When designing experiments to detect ERF6 phosphorylation:

• Sample preparation:

  • Include appropriate controls (non-phosphorylated samples)

  • Use phosphatase inhibitors during extraction to preserve phosphorylation status

  • Consider timing of sample collection (phosphorylation occurs rapidly after stress)

• Detection methodology:

  • Western blotting with ERF060 antibody will show band shifts for phosphorylated ERF6

  • Use Phos-tag™ gels for enhanced separation of phosphorylated forms

  • Consider dual antibody approach with anti-phospho-Ser/Thr antibodies

• Validation strategy:

  • Include constitutively active MKK4/MKK5 lines as positive controls

  • Use MPK3/MPK6 mutants as negative controls

  • Compare to in vitro phosphorylated recombinant ERF6 protein standards
    As demonstrated in previous research, phosphorylated ERF6 shows a characteristic band upshift in gel electrophoresis, particularly at Ser-266/Ser-269 residues, which can be abolished in mutants (ERF6 4A) .

What are the optimal conditions for immunoprecipitation using ERF060 antibody?

Based on protocols used in ERF6 research:

How can I validate the specificity of ERF060 antibody in my experimental system?

Validating specificity of ERF060 antibody requires a multi-step approach:

• Genetic validation:

  • Test on erf6 knockout/knockdown plants – should show absence or reduction of signal

  • Compare with overexpression lines – should show increased signal intensity

  • Use plants expressing tagged ERF6 (4myc-ERF6) to confirm co-localization with commercial antibody

• Biochemical validation:

  • Western blot analysis should reveal a single band at the expected molecular weight

  • Peptide competition assay using the immunizing peptide – should abolish signal

  • Cross-reactivity testing with closely related ERF family members

• Application-specific validation:

  • For immunohistochemistry: compare with mRNA expression patterns (in situ hybridization)

  • For ChIP assays: include negative control regions and IgG controls

  • For flow cytometry: follow standardized validation protocols as outlined in antibody validation literature
    Remember that validation should be performed for each specific application, as antibody performance can vary between techniques .

What quality control methods can detect degradation or loss of activity in ERF060 antibody?

To monitor antibody quality:

• Storage stability tests:

  • Regular immunoblotting of known positive samples using antibody aliquots

  • Monitor signal-to-noise ratio over time

  • Compare fresh vs. stored antibody performance

• Activity measurements:

  • ELISA against immobilized recombinant ERF6 protein

  • Comparative binding kinetics analysis

  • Thermal stability assessment using differential scanning fluorimetry

• Batch consistency monitoring:

  • Maintain reference standard from validated lot

  • Compare EC50 values across batches

  • Document lot-to-lot variation in detection sensitivity
    Establish internal quality control parameters with each new lot, including minimum signal threshold and maximum background levels for your specific experimental system.

Why might ERF060 antibody show weak or variable signal in Western blots?

Several factors can contribute to weak or variable ERF060 antibody signals:

• Sample-related issues:

  • Low abundance of ERF6 in unstimulated conditions – consider enrichment or using samples after stress treatment (ERF6 is upregulated after high light exposure or pathogen infection )

  • Rapid protein degradation – use fresh samples with protease inhibitors

  • Inefficient extraction – optimize buffer for nuclear proteins (ERF6 is a transcription factor)

• Technical considerations:

  • Insufficient transfer of high molecular weight proteins – adjust transfer conditions

  • Blocking reagent interference – test alternative blocking agents

  • Secondary antibody incompatibility – ensure proper species matching

• Biological variables:

  • Developmental stage-dependent expression – ERF6 shows temporal regulation

  • Tissue-specific expression patterns – select appropriate tissue samples

  • Stress-responsive expression – consider timing of sample collection after stress application
    When troubleshooting, systematically test each variable while keeping others constant, and include positive controls such as plants overexpressing ERF6 or tissues exposed to known inducers.

How can I optimize immunohistochemistry protocols for ERF060 antibody?

Optimization strategies for immunohistochemistry:

• Fixation optimization:

  • Test both cross-linking (paraformaldehyde) and precipitating (acetone) fixatives

  • Optimize fixation time (2-4 hours generally suitable)

  • Consider epitope retrieval methods if signal is weak

• Antibody conditions:

  • Perform titration series (1:100 to 1:2000) to determine optimal concentration

  • Test both short (2h room temperature) and long (overnight 4°C) incubation

  • Evaluate different diluents to reduce background

• Detection systems:

  • Compare direct fluorescent conjugates vs. multi-step amplification

  • Test tyramide signal amplification for low abundance targets

  • Optimize counterstaining to provide context while maintaining signal visibility
    Document all optimization steps and include appropriate controls (primary antibody omission, non-specific IgG, competing peptide) in each experiment.

How can I use ERF060 antibody to study the temporal dynamics of ERF6 activation in stress responses?

To study temporal dynamics of ERF6 activation:

• Time-course experimental design:

  • Collect samples at multiple timepoints after stress application

  • Focus on early timepoints (minutes to hours) based on known rapid responses

  • Include both phosphorylation status and total protein level analysis

• Advanced methodologies:

  • Combine with phospho-specific antibodies for multiplexed detection

  • Use cell fractionation to track nuclear translocation timing

  • Implement ChIP-seq to correlate ERF6 binding with target gene expression

• Quantitative analysis:

  • Develop a computational model of ERF6 activation kinetics

  • Calculate rate constants for activation/deactivation

  • Compare dynamics across different stressors or genetic backgrounds
    Previous research showed that ERF6 displayed a 4-fold upregulation at 10 and 30 min following high-light exposure, with strong decrease after 60 min , demonstrating the importance of capturing early timepoints in stress response studies.

What approaches can detect interactions between ERF6 and other components of the MPK3/MPK6 signaling pathway?

Several approaches can identify and characterize ERF6 interactions:

• Co-immunoprecipitation strategies:

  • Use ERF060 antibody as bait to pull down ERF6 and associated proteins

  • Reverse co-IP with antibodies against known or suspected interactors

  • Sequential IP (tandem affinity purification) for complex purification

• Proximity-based methods:

  • BioID/TurboID fusion proteins to identify proteins in ERF6 vicinity

  • FRET/BRET for real-time interaction monitoring in living cells

  • PLA (Proximity Ligation Assay) to visualize interactions in situ

• Functional interaction analysis:

  • ChIP-reChIP to identify co-occupancy at genomic loci

  • Sequential phosphorylation assays to determine order of modifications

  • Mutational analysis of interaction domains coupled with ERF060 immunoblotting
    Research has demonstrated that activated recombinant MPK3 and MPK6 strongly phosphorylate ERF6, while inactive forms show little activity , indicating direct interaction and functional relationship.

How can ERF060 antibody contribute to understanding the role of ERF6 in plant-pathogen interactions?

ERF060 antibody can provide crucial insights into plant-pathogen interactions:

• Infection time-course studies:

  • Monitor ERF6 levels and phosphorylation during different infection phases

  • Compare resistant vs. susceptible cultivars (as in the J11 vs. JL24 comparison)

  • Analyze ERF6 activation in response to different pathogen species or strains

• Spatial analysis of response:

  • Use immunohistochemistry to map ERF6 activation across infected tissues

  • Identify cell types with strongest responses

  • Monitor spreading of activation front relative to infection progression

• Integration with defense signaling networks:

  • Correlate ERF6 activation with defensin gene expression

  • Map ERF6-dependent transcriptional networks using ChIP-seq with ERF060

  • Analyze ERF6 activity in various defense signaling mutants
    Studies have shown that ERF6 is an important component of Arabidopsis defense against Botrytis cinerea, with phospho-mimicking mutants showing enhanced resistance and ERF6-EAR repressor forms exhibiting hypersusceptibility , demonstrating its functional significance in pathogen defense.

What considerations are important when developing custom modifications of ERF060 antibody for specialized applications?

When developing modified ERF060 antibodies:

• Conjugation chemistry selection:

  • Consider the nature of the label (fluorescent dye, enzyme, biotin)

  • Evaluate impact on antigen binding site (avoid modification near paratope)

  • Optimize conjugation ratio to maintain affinity while maximizing signal

• Validation of modified antibodies:

  • Compare binding kinetics before and after modification

  • Test specificity using knockout controls and competing antigens

  • Evaluate stability and shelf-life of the conjugate

• Application-specific optimization:

  • For FACS: optimize fluorophore brightness and stability

  • For super-resolution microscopy: consider photostability and quantum yield

  • For multiplexed assays: ensure spectral separation from other labels
    Recent advances in antibody engineering technologies, including those detailed in studies of engineered antibody Fc variants , can be adapted to enhance ERF060 antibody performance for specialized research applications.

How can I develop quantitative assays for ERF6 protein using ERF060 antibody?

Development of quantitative ERF6 assays:

• Sandwich ELISA development:

  • Use purified ERF6 protein to generate standard curves

  • Optimize capture and detection antibody concentrations

  • Validate assay parameters (limit of detection, linear range, reproducibility)

• Flow cytometry-based quantification:

  • Develop protocols similar to those used for JOVI.1 antibody quantification

  • Establish gates based on positive and negative control samples

  • Correlate signal intensity with known protein concentrations

• Automated image analysis:

  • Develop algorithms to quantify immunohistochemistry signal

  • Implement machine learning for cell-type specific quantification

  • Validate with orthogonal methods (Western blot, qPCR)
    When developing quantitative assays, ensure rigorous validation following guidelines similar to those used in clinical laboratories, including assessment of precision, accuracy, analytical sensitivity, and analytical specificity .

How could ERF060 antibody contribute to understanding transcriptional regulatory networks in plant stress responses?

ERF060 antibody can advance understanding of transcriptional networks through:

• ChIP-seq applications:

  • Map genome-wide ERF6 binding sites under different stress conditions

  • Identify condition-specific binding patterns

  • Correlate binding with transcriptional changes identified through RNA-seq

• Integrative multi-omics approaches:

  • Combine ERF6 ChIP-seq with phosphoproteomics and transcriptomics

  • Develop network models of ERF6-regulated processes

  • Identify feedback mechanisms and regulatory loops

• Single-cell applications:

  • Adapt techniques for single-cell protein detection using ERF060

  • Map cell-type specific ERF6 activation patterns

  • Correlate with single-cell transcriptomes
    Studies have shown that ERF6 regulates important defense genes, including PDF1.1 and PDF1.2, suggesting its role as a hub in coordinating defense responses . Expanding these findings with genome-wide approaches could reveal the full extent of ERF6 regulatory influence.

What emerging technologies could enhance the utility of ERF060 antibody in plant research?

Emerging technologies with potential applications:

• Advanced imaging approaches:

  • Super-resolution microscopy for subcellular localization

  • Live-cell imaging with membrane-permeable nanobody derivatives

  • Expansion microscopy for enhanced spatial resolution

• High-throughput screening applications:

  • Antibody-based protein arrays for ERF family comparative studies

  • Microfluidic antibody capture for single-cell protein quantification

  • Automated immunophenotyping in plant tissue sections

• Novel conjugation strategies:

  • PROTAC-antibody conjugates for targeted protein degradation studies

  • Photocaged antibody derivatives for spatiotemporal control

  • Bifunctional antibodies to study protein-protein interactions
    Recent advances in antibody engineering could be applied to ERF060 to enhance its capabilities for studying plant signaling dynamics and protein interactions with unprecedented precision.