ERF11 Antibody

What is ERF11 and why is it important in plant biology research?

ERF11 (also known as AtERF11) is a transcription factor belonging to the ERF (ETHYLENE RESPONSE FACTOR) subfamily VIII-B-1a of ERF/AP2 transcription factors in Arabidopsis thaliana. It contains an ERF/AP2 domain and a transcription repression EAR motif (DLNxxP) . ERF11 plays a critical role in promoting internode elongation through dual mechanisms: activating gibberellic acid (GA) biosynthesis and signaling pathways while simultaneously inhibiting ethylene biosynthesis .

The importance of ERF11 stems from its position as a molecular link between GA and ethylene pathways in modulating plant growth. Research has shown that overexpression of ERF11 results in increased plant height with longer internodes, while knockout mutants display shorter final height and internode length compared to wild-type plants . Understanding ERF11 function provides insight into fundamental plant growth regulation mechanisms.

How should I design controlled experiments to validate ERF11 antibody specificity?

When validating ERF11 antibody specificity, implement the following methodological approach:

• Positive and negative controls:

  • Use wild-type Arabidopsis samples as positive controls

  • Include erf11 knockout mutant samples as negative controls

  • Consider overexpression lines (ERF11-OE) for enhanced signal validation

• Cross-reactivity testing:

  • Test against closely related family members (ERF3, 4, 7-12) that share the ERF/AP2 domain

  • Include protein extracts from different tissues with varying ERF11 expression levels

• Epitope analysis:

  • Confirm epitope location relative to conserved domains (ERF/AP2 domain vs. EAR motif)

  • Consider potential post-translational modifications that might affect antibody binding

• Multiple detection methods:

  • Validate using at least two techniques (Western blot, immunoprecipitation, immunohistochemistry)

  • Include peptide competition assays to confirm binding specificity

Remember that ERF11 belongs to a subfamily with highly similar members, making specificity validation particularly important to avoid cross-reactivity with related proteins like ERF4 and ERF8 .

What are the recommended tissue types and extraction protocols for ERF11 detection?

For optimal ERF11 detection, focus on tissues with known expression and implement specialized extraction protocols:

Optimal tissue samples:

• Growing internodes (highest relevance for elongation studies)

• Stem tissue (where ERF11 function in height regulation is most evident)

• Seedling hypocotyl (for hypocotyl elongation assays)

Extraction protocol for nuclear transcription factors:

• Harvest fresh tissue and flash-freeze in liquid nitrogen

• Grind tissue to fine powder while maintaining freezing temperatures

• Extract using nuclear extraction buffer containing:

  • 50mM HEPES (pH 7.5)

  • 150mM NaCl

  • 1mM EDTA

  • 1% Triton X-100

  • 10% glycerol

  • Protease inhibitor cocktail

  • Phosphatase inhibitors (if studying phosphorylation states)

• Include 1mM DTT freshly added before use

• Centrifuge at 16,000×g at 4°C for 15 minutes

• Carefully separate nuclear fraction

• Verify protein integrity before antibody application

This specialized nuclear extraction protocol is essential because ERF11 functions as a transcription factor primarily localized in the nucleus, requiring careful preservation of nuclear proteins during extraction.

How can I use ERF11 antibodies to investigate protein-protein interactions with DELLA proteins?

To investigate ERF11-DELLA protein interactions, employ a multi-method approach:

Co-immunoprecipitation (Co-IP) strategy:

• Perform IP using anti-ERF11 antibody in plant tissue extracts

• Probe Western blots with anti-DELLA antibodies (particularly RGA)

• Include reciprocal Co-IP using anti-DELLA antibodies first, then probe for ERF11

• Use crosslinking agents (like DSP or formaldehyde) to stabilize transient interactions

• Include controls with GA treatments that degrade DELLA proteins

Experimental design considerations:

• Use both wild-type and genetic backgrounds with altered DELLA stability (e.g., rga-Δ17)

• Compare samples with and without GA treatment to manipulate DELLA levels

• Include erf11-1D samples where ERF11 is overexpressed

• Analyze protein complexes in different developmental stages

The research by Zhou et al. demonstrated that AtERF11 enhances GA signaling by antagonizing DELLA proteins through direct protein-protein interaction . Your experimental design should account for this interaction being potentially context-dependent and influenced by hormonal status.

What approaches can be used to quantify changes in ERF11 protein levels during plant hormone treatments?

For quantitative analysis of ERF11 protein dynamics during hormone treatments:

Quantitative Western blot protocol:

• Treat plant samples with hormones of interest (GA, ethylene, or precursors)

• Harvest tissues at multiple time points (0, 1, 3, 6, 12, 24 hours)

• Extract proteins using nuclear extraction protocol

• Perform Western blots with anti-ERF11 antibody

• Include loading controls (anti-histone H3 or other stable nuclear proteins)

• Use fluorescent secondary antibodies for linear quantification

• Image using a calibrated fluorescence scanner

• Analyze band intensities with appropriate software (ImageJ, etc.)

Data normalization approach:

• Normalize ERF11 signal to loading control

• Express as fold change relative to untreated samples

• Use biological and technical replicates (n ≥ 3)

• Apply appropriate statistical tests (ANOVA with post-hoc tests)

This approach allows for tracking ERF11 protein accumulation kinetics in response to hormones. Research has shown that ERF11 levels may be affected by both GA and ethylene pathways, making quantitative analysis essential for understanding regulatory mechanisms .

How can ERF11 antibodies be used to investigate transcriptional regulation of target genes?

For investigating ERF11's role in transcriptional regulation:

Chromatin Immunoprecipitation (ChIP) methodology:

• Cross-link proteins to DNA in plant tissues (preferably internodes or seedlings)

• Isolate and fragment chromatin

• Immunoprecipitate ERF11-bound DNA fragments using validated anti-ERF11 antibody

• Reverse cross-linking and purify DNA

• Analyze by qPCR for specific targets or ChIP-seq for genome-wide binding

Target gene selection strategy:

• Focus on GA biosynthesis genes (GA3ox1, GA20ox)

• Include ethylene biosynthesis genes (ACS2, ACS5)

• Add known targets of related ERF family members

• Consider genes with putative ERF binding elements in promoters

Control considerations:

• Input DNA (pre-immunoprecipitation) control

• IgG control for non-specific binding

• Positive control regions (known ERF11 binding sites)

• Negative control regions (non-target genes)

This approach will reveal direct binding targets of ERF11, helping elucidate its role in regulating GA biosynthesis genes and ethylene biosynthesis genes like ACS2 and ACS5, which ERF11 has been shown to repress .

What are the most common causes of non-specific binding with ERF11 antibodies and how can they be mitigated?

Non-specific binding is a significant challenge when using ERF11 antibodies due to conserved domains shared among ERF family members. Address these issues methodically:

Common causes and solutions for non-specific binding:

When troubleshooting, systematically test different blocking agents, antibody concentrations, and wash stringencies. Remember that ERF11 belongs to a subfamily with eight members (ERF3, 4, and 7–12), each containing an ERF/AP2 domain and a transcription repression EAR motif , making specificity particularly challenging.

How should I optimize immunohistochemistry protocols for detecting ERF11 in plant tissues?

For successful immunohistochemical detection of ERF11 in plant tissues:

Optimized fixation and embedding protocol:

• Fix fresh tissue in 4% paraformaldehyde in PBS (pH 7.4) for 4-6 hours at 4°C

• Perform gradual dehydration series (30%, 50%, 70%, 85%, 95%, 100% ethanol)

• Infiltrate and embed in a plant-specific embedding medium

Antigen retrieval considerations:

• Test citrate buffer (pH 6.0) heat-induced epitope retrieval

• Compare with enzymatic retrieval using proteinase K

• Optimize retrieval time based on tissue thickness

Immunostaining procedure:

• Block with 5% normal serum + 1% BSA in PBS for 1 hour

• Incubate with primary anti-ERF11 antibody (1:100-1:500 dilution) overnight at 4°C

• Wash extensively (6× 5 minutes) with PBS + 0.1% Tween-20

• Apply fluorescent or enzymatic secondary antibody (1:200-1:500) for 1-2 hours

• Counterstain nuclei (DAPI) and cell walls (calcofluor white)

• Mount in anti-fade medium

Critical controls:

• Omit primary antibody

• Use pre-immune serum

• Include erf11 knockout tissue sections

• Use competing peptide to confirm specificity

When analyzing results, focus on nuclear localization patterns since ERF11 functions as a transcription factor, with particular attention to internodal regions where its function in promoting elongation is most relevant .

What are the best approaches for generating and validating custom ERF11 antibodies?

For generating custom ERF11-specific antibodies:

Antigen design strategy:

• Select unique regions of ERF11 that differ from related ERF proteins

• Avoid the conserved ERF/AP2 domain and EAR motif shared among subfamily members

• Consider using:

  • Synthetic peptides from N or C-terminal regions (15-20 amino acids)

  • Recombinant partial proteins excluding conserved domains

  • Full-length ERF11 with subsequent cross-adsorption against related proteins

Production and purification approach:

• Immunize at least two rabbits for polyclonal production

• Consider monoclonal development for highest specificity

• Perform affinity purification against the immunizing antigen

• Include additional purification step against related ERF proteins

Comprehensive validation protocol:

This multi-parameter validation approach is essential because the antibody's utility in research applications will depend entirely on its specificity, particularly given the similarity between ERF11 and other ERF family members .

How should I analyze data from experiments investigating ERF11's role in gibberellic acid and ethylene signaling?

For analyzing ERF11's dual role in hormonal pathways:

Integrated data analysis framework:

• GA pathway analysis:

  • Measure bioactive GA levels (GA1, GA4) using LC-MS in wild-type vs. erf11 mutant vs. ERF11-OE

  • Quantify expression of GA biosynthesis genes (GA3ox1, GA20ox) by RT-qPCR

  • Assess hypocotyl/internode elongation in response to exogenous GA treatment

  • Measure DELLA protein levels (particularly RGA) by Western blot

• Ethylene pathway analysis:

  • Quantify ethylene production in internodes using gas chromatography

  • Measure expression of ACS2 and ACS5 genes by RT-qPCR

  • Assess triple response phenotypes to exogenous ethylene

  • Compare results across genetic backgrounds (wild-type, erf11, ERF11-OE)

• Integration model:

  • Normalize all data to appropriate controls

  • Plot time-course or dose-response curves

  • Perform correlation analysis between GA-related and ethylene-related parameters

  • Use multivariate statistics to identify relationships between pathways

Interpretation guidelines:

• Look for inverse correlations between ethylene production and GA levels

• Assess whether ERF11's effect on GA biosynthesis is dependent on its repression of ethylene biosynthesis

• Consider that ERF11 may have both direct effects (through protein-protein interactions with DELLAs) and indirect effects (through transcriptional regulation of hormone biosynthesis genes)

This integrated approach will help untangle the complex role of ERF11 as a dual regulator of both GA and ethylene pathways in controlling plant growth.

What statistical approaches are most appropriate for analyzing experiments using ERF11 antibodies?

When analyzing experiments employing ERF11 antibodies, select statistical methods based on the experimental design:

Recommended statistical approaches by experiment type:

Data visualization best practices:

• Present individual data points alongside means and error bars

• Use box plots or violin plots for distributions

• Include appropriate controls in all graphical representations

• Consider hierarchical clustering for multi-parameter experiments

Sample size determination:

• Perform power analysis based on preliminary data

• For Western blot quantification: minimum n=3 biological replicates

• For immunohistochemistry: minimum 5-10 sections per condition

• For ChIP experiments: minimum 3 biological replicates

These statistical approaches will ensure robust interpretation of ERF11 antibody experiments while accounting for the biological variability inherent in plant systems and the technical variability in antibody-based detection methods.

How can ERF11 antibodies be adapted for single-cell protein analysis in plant tissues?

Adapting ERF11 antibody techniques for single-cell resolution requires specialized approaches:

Single-cell protein detection strategies:

• High-resolution immunohistochemistry:

  • Use confocal or super-resolution microscopy

  • Implement tissue clearing techniques (ClearSee, TOMATO)

  • Apply spectral unmixing to distinguish ERF11 signal from autofluorescence

  • Quantify nuclear localization at single-cell level

• Flow cytometry with protoplasts:

  • Optimize protoplast isolation while preserving nuclear proteins

  • Fix and permeabilize cells gently

  • Use fluorescent anti-ERF11 antibodies

  • Include nuclear markers for co-detection

  • Sort cells based on ERF11 levels for downstream analysis

• Single-cell Western technologies:

  • Adapt microfluidic single-cell Western protocols for plant cells

  • Establish size standards specifically for ERF11 detection

  • Include cell-type specific markers

Data analysis considerations:

• Implement machine learning algorithms for automated cell classification

• Use dimensionality reduction techniques (t-SNE, UMAP) to identify cell populations

• Correlate ERF11 levels with cell morphological parameters (cell length, nuclear size)

• Create spatial maps of ERF11 distribution in developing tissues

This approach would be particularly valuable for understanding how ERF11's function in promoting internode elongation varies across different cell types within the stem, potentially revealing cell-specific responses to hormonal signals.

What are the latest approaches for studying the dynamics of ERF11-DELLA protein interactions in living plant cells?

For investigating ERF11-DELLA interaction dynamics in living cells:

Advanced live-cell imaging techniques:

• FRET-FLIM analysis:

  • Generate fluorescent protein fusions (ERF11-GFP, RGA-mCherry)

  • Measure Förster Resonance Energy Transfer using Fluorescence Lifetime Imaging

  • Quantify interaction by lifetime changes in donor fluorophore

  • Apply in different cell types and under various hormone treatments

• Split fluorescent protein complementation:

  • Create ERF11 and DELLA fusions with complementary fragments of fluorescent proteins

  • Visualize interaction through reconstituted fluorescence

  • Track spatial and temporal dynamics of interaction

  • Compare wild-type vs. mutant protein variants

• Optogenetic approaches:

  • Develop light-controllable ERF11 variants

  • Induce ERF11-DELLA interactions with precise spatiotemporal control

  • Monitor downstream responses to controlled interactions

  • Combine with hormone treatments to assess pathway integration

Quantitative analysis framework:

• Measure interaction kinetics (association/dissociation rates)

• Determine protein complex half-lives under different conditions

• Correlate interaction dynamics with downstream transcriptional responses

• Develop mathematical models of the ERF11-DELLA-hormone feedback loops

These approaches would extend the findings that "AtERF11 enhances GA signaling by antagonizing the function of DELLA proteins via direct protein-protein interaction" by revealing the dynamic nature of these interactions in living plant cells.

What are the emerging applications of ERF11 antibodies in understanding plant stress responses?

ERF11 antibodies have significant potential for elucidating stress response mechanisms:

Emerging research applications:

• Abiotic stress response studies:

  • Track ERF11 protein accumulation during drought, salt, and temperature stress

  • Correlate with changes in GA and ethylene signaling under stress conditions

  • Investigate post-translational modifications of ERF11 during stress adaptation

  • Compare stress responses across wild-type and erf11 mutant plants

• Developmental plasticity investigation:

  • Use ERF11 antibodies to map protein distribution during normal vs. stressed development

  • Quantify nuclear localization changes in response to environmental cues

  • Correlate with growth modulation under suboptimal conditions

• Hormone crosstalk visualization:

  • Implement multiplexed immunodetection of ERF11 alongside stress hormone markers

  • Create spatial maps of hormone response networks

  • Identify cell types where ERF11 mediates stress-growth tradeoffs

These applications build upon the established role of ERF11 in modulating both GA and ethylene pathways , potentially revealing how this dual regulatory function helps plants balance growth with stress adaptation, especially considering that ethylene is a major stress hormone while GA promotes growth processes that may be curtailed during stress.

What quality control standards should be implemented for ERF11 antibody usage in multi-laboratory research collaborations?

For consistent ERF11 antibody usage across laboratories:

Standardized quality control framework:

Implementation recommendations:

• Establish a central reference laboratory for antibody validation

• Distribute validated antibody aliquots from single preparations

• Create standardized positive control samples for inter-lab calibration

• Implement digital lab notebook templates for consistent documentation

• Develop shared analysis pipelines for comparable quantification

These standards ensure that differences in experimental outcomes across laboratories reflect biological variation rather than technical inconsistencies in antibody performance, particularly important given the challenges of specifically detecting ERF11 among its closely related family members .

How might ERF11 antibody research contribute to improving crop resilience and productivity?

ERF11 antibody research has translational potential for agriculture:

Translational research pathways:

• Crop improvement applications:

  • Use ERF11 antibodies to screen for natural variants with altered protein accumulation

  • Correlate ERF11 protein levels with desirable agronomic traits (stem strength, height)

  • Develop rapid screening tools for breeding programs

• Targeted genetic modification guidance:

  • Employ ERF11 antibodies to validate engineered variants in crop species

  • Monitor protein expression patterns in transgenic lines

  • Assess protein-level responses to environmental variables

• Stress resilience mechanisms:

  • Map ERF11 protein dynamics during drought, flooding, and temperature stress

  • Identify conditions where ERF11 mediates adaptive growth responses

  • Develop markers for selecting varieties with optimized ERF11 regulation

Potential agricultural impacts:

• Development of semi-dwarf varieties with optimized ERF11 activity

• Improved lodging resistance through modulated stem growth

• Enhanced resilience to climate variability

• Balanced growth-defense responses for sustainable yield

This translational potential builds on the fundamental finding that ERF11 promotes internode elongation , suggesting that fine-tuning its expression or activity could lead to crops with optimized height, improved stem strength, and better adaptation to environmental stress.