ERF086 Antibody

What is ERF086 and why is it significant in plant research?

ERF086 is an ethylene-responsive transcription factor belonging to the AP2/ERF family that plays crucial roles in plant stress responses and development. This protein is particularly significant because it mediates plant responses to various environmental stressors including drought, salinity, and pathogen attack through the ethylene signaling pathway . The study of ERF086 contributes to understanding transcriptional regulation during plant stress adaptation, which has implications for crop improvement and agricultural sustainability.

How is ERF086 antibody validated for specificity in plant tissue samples?

Validation of ERF086 antibody specificity involves multiple complementary approaches:

• Western blot analysis: Using recombinant ERF086 protein as a positive control alongside plant tissue extracts to confirm the antibody detects a single band of the expected molecular weight (typically 25-30 kDa for ERF family proteins).

• Immunoprecipitation followed by mass spectrometry: To confirm that the antibody pulls down ERF086 specifically from plant lysates.

• Comparative analysis with knockout/knockdown plants: Demonstrating reduced or absent signal in plants where ERF086 expression has been genetically suppressed.

• Cross-reactivity testing: Evaluating potential cross-reactivity with other ERF family members through comparison with recombinant proteins .

Complete validation should demonstrate consistent reactivity across multiple experimental conditions with minimal background signal.

What are the optimal sample preparation methods for detecting ERF086 in plant tissues?

For nuclear proteins like ERF086, nuclear extraction protocols typically yield better results than whole cell lysates. Addition of phosphatase inhibitors is recommended when studying post-translational modifications .

How should researchers optimize immunodetection protocols specifically for ERF086 antibody?

Optimizing immunodetection for ERF086 requires careful consideration of several technical parameters:

For Western blot applications:

• Use PVDF membranes rather than nitrocellulose for better protein retention

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

• Dilute primary ERF086 antibody to 1:1000-1:2000 in blocking buffer

• Incubate overnight at 4°C with gentle agitation

• Include extensive washing steps (5 × 5 minutes with TBST)

• Optimize secondary antibody concentration (typically 1:5000-1:10000)

• Consider enhanced chemiluminescence detection for higher sensitivity

For immunohistochemistry:

• Fixation with 4% paraformaldehyde is preferable to methanol-based fixatives

• Perform antigen retrieval (citrate buffer, pH 6.0, 95°C for 15 minutes)

• Use longer primary antibody incubation times (overnight at 4°C)

• Include proper negative controls (secondary antibody only, pre-immune serum)

What are the critical differences in experimental design when studying ERF086 under different stress conditions?

When studying ERF086 under different stress conditions, several critical experimental design factors must be considered:

Temporal dynamics:

• Ethylene-responsive transcription factors like ERF086 show biphasic responses

• Early sampling (30 min-2 hours) captures immediate transcriptional activation

• Later sampling (12-48 hours) reveals sustained regulatory networks

Stress-specific considerations:

• For drought stress: Implement controlled water withholding with soil moisture monitoring

• For salt stress: Use standardized NaCl concentrations (typically 100-200 mM) with graduated exposure

• For pathogen stress: Synchronize infection timing and quantify pathogen load

Control groups:

• Include both negative controls (unstressed plants) and positive controls (plants treated with ethylene or ethephon)

• Match developmental stages precisely between treatment and control groups

Data normalization:

• Use multiple reference genes (e.g., ACTIN2, UBIQUITIN10, EF1α) for qPCR normalization

• Quantify total protein loading using standardized methods like Ponceau S staining for Western blots

This comparative approach enables distinguishing stress-specific from general stress responses mediated by ERF086 .

What are the most reliable co-immunoprecipitation protocols for studying ERF086 protein interactions?

For studying ERF086 protein interactions via co-immunoprecipitation, the following protocol adaptations provide reliable results:

• Cross-linking step: Implement in vivo crosslinking with 1% formaldehyde for 10 minutes before tissue harvesting to preserve transient interactions common in transcription factor complexes.

• Buffer optimization: Use a nuclear extraction buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% NP-40, supplemented with protease inhibitors, phosphatase inhibitors, and 1 mM DTT.

• Pre-clearing step: Pre-clear lysates with protein A/G beads conjugated to non-specific IgG for 1 hour at 4°C.

• Antibody coupling: Covalently couple purified ERF086 antibody to protein A/G beads using dimethyl pimelimidate to prevent antibody leaching.

• Washing stringency gradient: Implement sequential washes with increasing stringency to minimize false positives.

• Elution conditions: Use a gentle elution with peptide competition rather than harsh denaturing conditions.

• Validation controls: Include IgG control, input sample, and where possible, samples from ERF086-knockout plants.

This approach has been shown to reliably identify components of transcription factor complexes while minimizing false positives from non-specific binding .

How can researchers accurately differentiate between ERF086 activity and that of closely related ERF family members?

Differentiating ERF086 activity from other closely related ERF family members requires a multi-faceted approach:

Antibody specificity validation:

• Perform epitope mapping to identify antibody binding regions

• Test cross-reactivity against recombinant proteins of closely related ERF family members

• Use peptide competition assays with unique peptide sequences from the ERF086 protein

Genetic approaches:

• Employ CRISPR/Cas9-mediated knockout specifically targeting ERF086

• Utilize RNAi with carefully designed sequences targeting unique regions of ERF086 transcript

• Perform complementation studies with ERF086 variants to confirm specificity

Bioinformatic analysis:

• Conduct motif enrichment analysis for ERF086-specific binding motifs in target promoters

• Compare ChIP-seq data between ERF086 and related family members

• Analyze protein-protein interaction networks specific to ERF086

Functional readouts:

• Design reporter assays with promoters specifically regulated by ERF086

• Analyze transcriptional outputs using RNA-seq in ERF086 mutant backgrounds

This integrated approach helps establish ERF086-specific functions within the broader ERF family context .

What statistical approaches are most appropriate for analyzing antibody-based detection of ERF086 across different experimental conditions?

For robust statistical analysis of ERF086 antibody-based detection across experimental conditions, the following approaches are recommended:

For Western blot densitometry:

• Apply log-transformation to achieve normality for parametric testing

• Use ANOVA with Tukey's post-hoc test for multiple condition comparisons

• Implement repeated measures designs when comparing the same samples under different conditions

• Calculate coefficient of variation across technical replicates (should be <15%)

For immunohistochemistry quantification:

• Use image analysis software with consistent thresholding parameters

• Employ mixed-effects models that account for both technical and biological variation

• Consider non-parametric tests (e.g., Mann-Whitney U) when normality cannot be assumed

For ELISA data:

• Fit four-parameter logistic regression curves for standard curves

• Calculate lower limit of detection as 3× standard deviation of blank

• Use analysis of covariance (ANCOVA) when comparing across plates

Advanced considerations:

• Apply finite mixture models based on scale mixtures of Skew-Normal distributions for complex antibody data patterns

• Implement Bayesian hierarchical models to account for nested experimental designs

Complete statistical analysis should include sample size calculations, power analysis, and transparent reporting of all exclusion criteria .

What are the known contradictions in ERF086 research literature and how can they be reconciled?

Several noteworthy contradictions exist in the ERF086 research literature:

Subcellular localization discrepancies:
Some studies report exclusive nuclear localization of ERF086, while others observe cytoplasmic retention under certain conditions. This contradiction can be reconciled by considering:

• Cell-type specific regulation of nuclear import/export

• Stress-dependent phosphorylation affecting localization

• Differences in detection methods (fixed vs. live imaging)

• Presence of alternative splice variants with different localization patterns

Transcriptional activity conflicts:
ERF086 has been reported as both an activator and repressor of transcription depending on the study. This apparent contradiction is likely due to:

• Promoter context dependency (presence of co-factors)

• Post-translational modifications altering activity

• Concentration-dependent effects (activation at low levels, repression at high levels)

• Developmental stage-specific roles

Species-specific functional differences:
Functional studies in tomato and cotton show different physiological roles for ERF086. This can be reconciled through:

• Phylogenetic analysis of sequence divergence

• Domain swap experiments to identify functional differences

• Comparative genomics of target gene repertoires

• Analysis of species-specific protein interaction networks

Methodologically, these contradictions can be addressed through comprehensive experimental designs that directly compare conditions under standardized protocols .

How can ERF086 antibody be effectively used in chromatin immunoprecipitation (ChIP) experiments to identify direct target genes?

For effective ChIP experiments using ERF086 antibody, researchers should implement the following protocol modifications:

Chromatin preparation:

• Use dual crosslinking (1% formaldehyde followed by EGS/DSG) to improve capture of transcription factor interactions

• Optimize sonication conditions for plant tissues (typically 12-15 cycles of 30s ON/30s OFF) to achieve 200-500bp fragments

• Verify sonication efficiency via gel electrophoresis before proceeding

• Pre-clear chromatin with protein A/G beads conjugated to non-specific IgG

Antibody considerations:

• Validate antibody specifically for ChIP applications using known ERF086 binding sites

• Use 3-5μg of purified ERF086 antibody per immunoprecipitation

• Include spike-in controls with recombinant ERF086 protein bound to known DNA sequences

• Perform sequential ChIP (re-ChIP) for identifying complex regulatory modules

Controls and normalization:

• Include input (non-immunoprecipitated) chromatin as quantification control

• Use IgG negative control to establish background enrichment

• Implement spike-in normalization with exogenous chromatin (e.g., Drosophila chromatin)

• Include positive controls (regions known to be bound by ERF086) and negative controls (non-bound regions)

Analysis recommendations:

• Analyze enrichment using both candidate gene approaches (qPCR) and genome-wide methods (ChIP-seq)

• For ChIP-seq, use specialized peak callers optimized for transcription factors (e.g., MACS2)

• Perform motif enrichment analysis on identified binding regions

• Integrate with RNA-seq data to correlate binding with expression changes

This comprehensive approach enables reliable identification of direct ERF086 target genes and regulatory networks .

What are the methodological considerations for developing phospho-specific antibodies against ERF086?

Developing phospho-specific antibodies against ERF086 requires careful attention to several methodological considerations:

Phosphorylation site selection:

• Conduct bioinformatic analysis to identify conserved phosphorylation motifs in ERF086

• Prioritize sites with documented biological relevance (e.g., affecting DNA binding or protein stability)

• Consider sites with known kinase recognition motifs (e.g., MAPK or CDK targets)

• Select sites with favorable surrounding amino acid sequences for immunogenicity

Peptide design strategy:

• Design 12-15 amino acid peptides containing the phosphorylated residue at a central position

• Include carrier protein conjugation (typically KLH) for enhanced immunogenicity

• Synthesize both phosphorylated and non-phosphorylated peptides for screening and purification

• Conduct quality control via mass spectrometry to confirm phosphorylation status

Immunization and screening protocol:

• Implement a multiple-host strategy (typically 2-4 rabbits per epitope)

• Use adjuvants specifically optimized for phospho-epitopes

• Design a screening protocol with phospho- and non-phospho peptide ELISA

• Establish stringent selection criteria (>10:1 selectivity for phospho-forms)

Purification methodology:

• Perform tandem affinity purification using both negative selection (non-phosphopeptide column) and positive selection (phosphopeptide column)

• Validate purified antibodies via Western blot with phosphatase-treated controls

• Confirm specificity using phosphomimetic mutants (S/T→D/E) and phospho-null mutants (S/T→A)

• Test antibody performance across multiple experimental conditions (e.g., different stress treatments)

These methodological considerations help ensure development of highly specific phospho-ERF086 antibodies for studying post-translational regulation mechanisms .

How can researchers apply advanced structural biology techniques to better understand ERF086 antibody binding mechanisms?

Applying advanced structural biology techniques to understand ERF086 antibody binding mechanisms requires integration of multiple approaches:

X-ray crystallography:

• Co-crystallize Fab fragments of ERF086 antibody with target epitope peptides

• Use molecular replacement with known antibody structures as search models

• Optimize crystallization conditions with additives that stabilize antibody-antigen complexes

• Analyze binding interfaces at atomic resolution to identify key interaction residues

Cryo-electron microscopy (cryo-EM):

• Apply single-particle cryo-EM for studying full-length ERF086-antibody complexes

• Implement computational particle sorting to handle conformational heterogeneity

• Perform focused classification on binding interface regions

• Generate 3D reconstructions at sub-4Å resolution to visualize binding determinants

Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

• Map conformational changes in both antibody and ERF086 upon binding

• Identify regions with altered solvent accessibility

• Compare binding dynamics across different antibody variants

• Correlate HDX protection patterns with functional epitopes

Computational modeling:

• Generate synthetic antibody-antigen structures using platforms like Absolut!

• Apply molecular dynamics simulations to study binding kinetics and stability

• Use machine learning approaches to predict binding affinities and cross-reactivity

• Implement structure-based epitope mapping to guide antibody engineering

Integration with functional data:

• Correlate structural insights with functional assays (e.g., competitive binding)

• Design structure-guided mutations to validate key interaction residues

• Apply alanine-scanning mutagenesis to quantify energetic contributions

• Develop structure-based screening assays for next-generation antibody development

This multi-technique approach provides comprehensive understanding of ERF086 antibody binding mechanisms, enabling rational improvement of antibody specificity and affinity .

How might single-cell technologies be applied to study ERF086 localization and activity in heterogeneous plant tissues?

Single-cell technologies offer unprecedented opportunities for studying ERF086 in complex plant tissues:

Single-cell immunohistochemistry innovations:

• Adapt tissue clearing protocols (e.g., ClearSee) specifically for plant tissues to improve antibody penetration

• Implement tyramide signal amplification for detecting low-abundance ERF086

• Develop multi-epitope ligand cartography techniques to map ERF086 co-localization with interaction partners

• Apply expansion microscopy to improve spatial resolution of ERF086 localization

Single-cell genomics approaches:

• Adapt single-cell RNA-seq protocols for plant protoplasts to correlate ERF086 activity with transcriptional outputs

• Implement single-cell ATAC-seq to map chromatin accessibility at ERF086 binding sites

• Develop plant-specific CUT&Tag or CUT&Run protocols to map ERF086 binding at single-cell resolution

• Apply single-cell proteomics techniques to quantify ERF086 protein abundance across cell types

Spatial transcriptomics integration:

• Correlate spatial transcriptomics data with ERF086 immunolocalization

• Develop in situ sequencing methods compatible with plant tissues

• Implement multiplexed FISH techniques to simultaneously visualize ERF086 and target transcripts

• Create computational frameworks to integrate spatial and single-cell data

Technical challenges and solutions:

• Adapt cell isolation protocols to preserve native transcription factor complexes

• Develop specialized fixation methods that maintain both protein localization and RNA integrity

• Implement microfluidic approaches for handling small populations of plant cells

• Create data integration pipelines specifically for plant single-cell multi-omics

These emerging technologies will enable unprecedented insights into cell-type-specific ERF086 functions in complex plant tissues .

What are the potential applications of nanobody technology for studying ERF086 in live plant cells?

Nanobody technology offers several advantages for studying ERF086 in live plant cells:

Development strategies:

• Immunize camelids (e.g., llamas) with purified recombinant ERF086 protein

• Screen nanobody libraries using phage display against native conformation ERF086

• Perform epitope binning to identify nanobodies targeting different ERF086 domains

• Engineer nanobody-based biosensors by fusion with fluorescent proteins

Live-cell imaging applications:

• Generate fluorescently-tagged anti-ERF086 nanobodies for real-time tracking

• Develop FRET-based nanobody pairs to monitor ERF086 conformational changes

• Create bimolecular fluorescence complementation (BiFC) systems with nanobodies to visualize ERF086 interactions

• Implement optogenetic nanobody tools to manipulate ERF086 localization with light

Functional manipulation:

• Design nanobodies that specifically block ERF086 DNA binding domains

• Create nanobody-based degradation systems (e.g., nanobody-auxin degrons) for rapid ERF086 depletion

• Develop nanobody-based synthetic transcription factors to redirect ERF086 activity

• Implement nanobody-based chromatin recruitment tools to artificially target ERF086

Technical advantages for plant systems:

• Smaller size (15 kDa) enables better tissue penetration than conventional antibodies

• High stability allows functionality in various cellular compartments

• Single-domain nature facilitates expression as intrabodies in plant cells

• Reduced immunogenicity when expressed in planta

These nanobody applications would enable unprecedented spatiotemporal manipulation and visualization of ERF086 in living plant systems .

How could antibody engineering approaches be applied to create multi-specific antibodies for simultaneous detection of ERF086 and related stress response factors?

Advanced antibody engineering approaches can create multi-specific detection systems for ERF086 and related factors:

Bispecific antibody formats:

• DVD-Ig (dual-variable domain immunoglobulin) format with ERF086 specificity in one arm and related factor specificity in the other

• Tandem scFv constructs linking anti-ERF086 single-chain variable fragments with complementary specificities

• Knobs-into-holes heterodimeric antibodies with dual specificities

• CrossMAb technology to ensure proper light chain pairing in bispecific formats

Trispecific antibody engineering:

• Adapt trispecific antibody frameworks (as used in HIV research) for plant transcription factor detection

• Implement "hub-and-spoke" designs with ERF086 binding at the hub and related factors at the spokes

• Develop sequential binding-optimized designs to detect transcription factor complexes

• Create asymmetric trispecific formats with optimized binding kinetics for each target

Optimization strategies:

• Use yeast or phage display to screen for optimal domain combinations

• Implement alanine scanning mutagenesis to minimize interference between binding sites

• Apply computational design to optimize linker length and composition

• Conduct in vitro evolution to enhance specificity and affinity

Validation methodology:

• Develop multi-color immunofluorescence systems to validate simultaneous binding

• Create surface plasmon resonance (SPR) assays to measure binding to individual and combined antigens

• Implement bio-layer interferometry for real-time binding kinetics analysis

• Design sandwich ELISA formats to validate cooperative binding

These engineered multi-specific antibodies would enable simultaneous monitoring of complex transcriptional networks involving ERF086 and related factors in plant stress responses .