ERF118 Antibody

What is ERF118 and why is it important in plant research?

ERF118 (Ethylene-responsive transcription factor 118) belongs to the ERF family of transcription factors that regulate various developmental processes and stress responses in plants. It plays a significant role in ethylene signaling pathways, which are crucial for plant growth regulation, stress adaptation, and adventitious root formation. Research indicates that ERF118 is involved in gene expression changes during adventitious root development and may interact with other hormone signaling pathways, particularly IAA (indole-3-acetic acid) . Understanding ERF118 function can provide insights into plant developmental biology and stress responses, making it an important target for agricultural biotechnology research.

What sample types can be analyzed using ERF118 antibodies?

ERF118 antibodies can be used to detect the protein in various plant tissue samples, particularly from model organisms like Arabidopsis thaliana. Suitable sample types include:

• Plant tissue lysates (roots, stems, leaves, flowers)

• Cultured plant cells

• Protoplasts

• In vitro regenerated tissues

• Transgenic plant materials expressing tagged versions of ERF118
  Similar to other plant protein antibodies, ERF118 antibodies would typically be validated for specific applications such as Western blotting, immunohistochemistry, and immunofluorescence in plant tissues .

How do ERF118 expression patterns differ across plant tissues and developmental stages?

ERF118 expression exhibits organ-specific characteristics and can be influenced by various environmental and hormonal factors. Research on related ethylene-responsive transcription factors indicates that these proteins often show differential expression patterns during development. For example, studies of similar ERF family members in lotus (Nelumbo nucifera) demonstrate that expression can be induced by sucrose and indoleacetic acid (IAA) . Expression analyses using ERF118 antibodies can help monitor protein levels across different tissues (roots, stems, leaves) and developmental stages to correlate expression patterns with specific physiological processes or stress responses.

What are the optimal conditions for Western blot detection of ERF118?

For optimal Western blot detection of ERF118 in plant samples:

• Sample preparation:

  • Homogenize plant tissue in extraction buffer containing protease inhibitors

  • Use 10-20 μg of total protein per lane

• Gel electrophoresis:

  • Separate proteins on 10-12% SDS-PAGE gels

  • Use appropriate molecular weight markers (ERF118 is approximately 25-30 kDa)

• Transfer and blocking:

  • Transfer to PVDF membrane at 100V for 1 hour

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

• Antibody incubation:

  • Primary antibody: Dilute ERF118 antibody at 1:1000 in blocking solution

  • Incubate overnight at 4°C with gentle agitation

  • Secondary antibody: Use appropriate HRP-conjugated secondary at 1:2000-1:5000

• Detection:

  • Develop using ECL detection system

  • Expected band size will depend on the specific ERF118 variant (typically 25-30 kDa)

How can I optimize immunohistochemistry protocols for ERF118 detection in plant tissues?

For successful immunohistochemical detection of ERF118 in plant tissues:

• Tissue fixation and processing:

  • Fix tissues in 4% paraformaldehyde

  • Embed in paraffin or prepare frozen sections

  • Section at 5-10 μm thickness

• Antigen retrieval:

  • Perform heat-mediated antigen retrieval with citrate buffer (pH 6.0)

  • Boil sections for 10-20 minutes, then cool to room temperature

• Blocking and antibody incubation:

  • Block with 5% normal serum in PBS with 0.1% Triton X-100

  • Incubate with ERF118 antibody at 1:100 dilution overnight at 4°C

  • Wash thoroughly with PBS (3×5 minutes)

  • Incubate with appropriate secondary antibody for 1-2 hours at room temperature

• Visualization:

  • Develop using DAB substrate for brightfield microscopy

  • For fluorescence, use appropriate fluorophore-conjugated secondary antibodies

  • Counterstain nuclei with DAPI if using fluorescence

• Controls:

  • Include negative controls (omitting primary antibody)

  • Use tissues from ERF118 knockout plants as negative controls when available

What are the best co-immunoprecipitation approaches for studying ERF118 interactions with other proteins?

To investigate ERF118 protein-protein interactions:

• Sample preparation:

  • Homogenize plant tissue in non-denaturing lysis buffer

  • Include protease inhibitors, phosphatase inhibitors, and mild detergents

• Pre-clearing:

  • Incubate lysate with protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation to reduce non-specific binding

• Immunoprecipitation:

  • Add ERF118 antibody (2-5 μg) to pre-cleared lysate

  • Incubate overnight at 4°C with rotation

  • Add fresh protein A/G beads and incubate for 2-4 hours

  • Wash beads 4-5 times with washing buffer

• Elution and analysis:

  • Elute proteins by boiling in Laemmli buffer

  • Analyze by SDS-PAGE followed by Western blot or mass spectrometry

• Controls:

  • Use IgG from the same species as a negative control

  • Include input samples (pre-immunoprecipitation lysate)

  • Consider reverse co-IP using antibodies against suspected interacting partners
    This approach allows identification of proteins that interact with ERF118, potentially revealing its role in transcriptional complexes and signaling pathways.

How can I use ChIP-seq with ERF118 antibodies to identify genome-wide binding sites?

For ChIP-seq analysis of ERF118 binding sites:

• Sample preparation:

  • Cross-link plant tissue with 1% formaldehyde for 10 minutes

  • Quench with 0.125 M glycine

  • Extract and sonicate chromatin to 200-500 bp fragments

• Immunoprecipitation:

  • Incubate sonicated chromatin with ERF118 antibody (5-10 μg)

  • Include appropriate controls (IgG, input DNA)

  • Capture antibody-chromatin complexes with protein A/G beads

  • Wash thoroughly to remove non-specific binding

• DNA recovery and library preparation:

  • Reverse cross-links and purify DNA

  • Prepare sequencing libraries according to platform specifications

  • Include appropriate adapter ligation and PCR amplification steps

• Sequencing and data analysis:

  • Perform high-throughput sequencing (minimum 10 million reads)

  • Align reads to reference genome

  • Identify enriched regions using peak-calling algorithms

  • Perform motif analysis to identify ERF118 binding motifs

  • Integrate with RNA-seq data to correlate binding with gene expression
    This method will reveal genomic regions bound by ERF118, potentially identifying direct target genes involved in ethylene responses and developmental processes in plants.

What strategies can I use to investigate ERF118 phosphorylation status and its effect on function?

To study ERF118 phosphorylation:

• Phosphorylation detection:

  • Immunoprecipitate ERF118 using specific antibodies

  • Analyze by Western blot using phospho-specific antibodies (if available)

  • Alternatively, use general phospho-serine/threonine/tyrosine antibodies

  • Consider Phos-tag™ SDS-PAGE to separate phosphorylated from non-phosphorylated forms

• Mass spectrometry analysis:

  • Immunoprecipitate ERF118

  • Perform in-gel digestion

  • Analyze by LC-MS/MS with phosphopeptide enrichment

  • Map phosphorylation sites to protein sequence

• Functional analysis:

  • Generate phospho-mimetic (S/T→D/E) and phospho-null (S/T→A) mutants

  • Express mutants in ERF118-deficient plants

  • Compare phenotypes and transcriptional activity

  • Perform DNA binding assays to determine effects on target recognition

• Kinase identification:

  • Use kinase inhibitors to identify candidate kinase families

  • Perform in vitro kinase assays with purified candidates

  • Validate interactions in planta using BiFC or co-IP
    This approach would reveal how phosphorylation regulates ERF118 activity in response to ethylene or environmental stresses, providing insights into post-translational regulation of plant transcription factors .

Why might I see non-specific binding when using ERF118 antibodies in Western blots?

Non-specific binding in ERF118 Western blots can occur for several reasons:

• Antibody quality issues:

  • Solution: Validate antibody using positive controls (recombinant ERF118) and negative controls (ERF118 knockout samples)

  • Test multiple antibody concentrations (1:500, 1:1000, 1:2000) to optimize signal-to-noise ratio

• Cross-reactivity with related ERF family members:

  • Solution: Increase washing stringency (higher salt concentration in wash buffers)

  • Use more diluted primary antibody

  • Pre-adsorb antibody with plant lysates from ERF118 knockout plants

• Sample preparation issues:

  • Solution: Ensure complete protein denaturation

  • Add protease inhibitors freshly before extraction

  • Avoid sample overloading

• Blocking inadequacies:

  • Solution: Test alternative blocking agents (BSA vs. milk)

  • Increase blocking time (2 hours at room temperature)

  • Add 0.1% Tween-20 to blocking and antibody solutions

• Detection sensitivity:

  • Solution: Use enhanced chemiluminescence substrates for low abundance proteins

  • Consider using fluorescent secondary antibodies for cleaner background

How can I differentiate between ERF118 and other closely related ERF family members?

Differentiating between ERF118 and related ERF proteins requires careful experimental design:

• Antibody selection:

  • Use antibodies raised against unique regions of ERF118

  • Consider using peptide antibodies targeting non-conserved domains

  • Validate specificity using recombinant proteins of multiple ERF family members

• Molecular techniques:

  • Use tagged versions of ERF118 (e.g., GFP, FLAG, HA) in transgenic plants

  • Perform siRNA/CRISPR knockdown of ERF118 to confirm antibody specificity

  • Design PCR primers targeting unique regions for transcript verification

• Analytical approaches:

  • Use high-resolution SDS-PAGE to separate closely related ERFs by size

  • Consider 2D gel electrophoresis to separate by both size and charge

  • Perform mass spectrometry analysis to identify peptides unique to ERF118

• Expression pattern analysis:

  • Compare tissue and developmental expression patterns of ERF118 vs. related ERFs

  • Analyze responses to specific stimuli that may differentially regulate family members
    This multi-faceted approach will help ensure that you're specifically detecting ERF118 and not related family members, which is critical for accurate interpretation of results .

What are the best preservation methods for maintaining ERF118 epitope integrity in plant samples?

To preserve ERF118 epitope integrity:

• Fixation methods:

  • For immunohistochemistry: Use 4% paraformaldehyde (optimal for preserving protein structure)

  • Avoid over-fixation, which can mask epitopes

  • For cryosectioning: Flash freezing in liquid nitrogen followed by embedding in OCT compound

• Protein extraction:

  • Extract proteins in buffers containing protease inhibitors

  • Include reducing agents (DTT or β-mercaptoethanol) to preserve disulfide bonds

  • Extract at 4°C to minimize degradation

• Storage considerations:

  • Store tissue samples at -80°C for long-term preservation

  • For protein extracts, add glycerol (10-20%) before freezing

  • Avoid repeated freeze-thaw cycles

• Antigen retrieval:

  • For fixed tissues, optimize antigen retrieval methods (citrate buffer, pH 6.0)

  • Test enzymatic retrieval versus heat-induced retrieval

  • Adjust retrieval times based on fixation duration

• Sample processing:

  • Process samples quickly to minimize degradation

  • Keep samples cold throughout processing

  • Consider using vacuum infiltration for larger tissue samples to ensure complete fixative penetration
    These methods will help maintain the structural integrity of ERF118 epitopes, ensuring better antibody recognition and more reliable experimental results.

How should I quantify and normalize ERF118 expression data from Western blots?

For accurate quantification of ERF118 from Western blots:

• Image acquisition:

  • Capture images within the linear range of the detection system

  • Avoid saturation of signal

  • Include a dilution series of a positive control for standard curve generation

• Normalization approaches:

  • Use housekeeping proteins (e.g., actin, tubulin, GAPDH) as loading controls

  • Consider total protein normalization methods (Ponceau S, SYPRO Ruby)

  • For plant samples, RuBisCO large subunit can serve as a loading control

• Quantification methods:

  • Use densitometry software (ImageJ, Image Lab, etc.)

  • Measure integrated density of ERF118 bands

  • Subtract background from an adjacent area

  • Calculate relative expression as ratio of ERF118/loading control

• Statistical analysis:

  • Perform experiments with at least three biological replicates

  • Apply appropriate statistical tests (t-test, ANOVA) based on experimental design

  • Report means with standard error or standard deviation

• Presentation guidelines:

  • Present representative blot images alongside quantification

  • Include molecular weight markers

  • Use consistent contrast adjustments across compared samples
    This approach ensures accurate, reproducible quantification of ERF118 protein levels across different experimental conditions .

What considerations are important when interpreting co-localization data of ERF118 with other proteins?

When interpreting co-localization data:

• Technical considerations:

  • Ensure proper controls for antibody specificity

  • Use appropriate fluorophore combinations to minimize spectral overlap

  • Apply consistent imaging parameters across samples

  • Consider super-resolution microscopy for closely associated proteins

• Quantitative analysis:

  • Calculate co-localization coefficients (Pearson's, Manders')

  • Use automated co-localization software to reduce bias

  • Establish threshold values based on control experiments

  • Analyze multiple cells across multiple fields

• Biological interpretation:

  • Co-localization does not necessarily indicate direct interaction

  • Consider sub-cellular compartment size relative to resolution limits

  • Correlate with functional assays (FRET, BiFC, co-IP)

  • Consider temporal dynamics of interactions

• Common pitfalls:

  • Chromatic aberration can create false co-localization

  • Fixation artifacts may alter protein localization

  • Overexpression can cause non-physiological localization

  • Z-axis limitations can create false positives in projected images

• Validation approaches:

  • Confirm co-localization using orthogonal methods

  • Use proximity ligation assays for improved specificity

  • Perform time-course experiments to capture dynamic interactions
    These considerations will help avoid misinterpretation of co-localization data and provide stronger evidence for genuine physiological interactions with ERF118.

How can I integrate ERF118 protein expression data with transcriptomic datasets?

To integrate ERF118 protein data with transcriptomics:

• Data preparation:

  • Normalize protein expression data from Western blots or proteomics

  • Process RNA-seq data using standard pipelines (quality control, alignment, quantification)

  • Ensure comparable experimental conditions and time points

• Correlation analysis:

  • Calculate correlation coefficients between ERF118 protein levels and mRNA expression

  • Perform time-lag analysis to account for delays between transcription and translation

  • Generate scatter plots with regression analysis

• Pathway integration:

  • Map ERF118 and its transcriptional targets to known signaling pathways

  • Perform gene set enrichment analysis (GSEA) on genes correlated with ERF118 protein levels

  • Use tools like Cytoscape to visualize integrated networks

• Multi-omics approaches:

  • Consider additional datasets (e.g., ChIP-seq, phosphoproteomics)

  • Use tools designed for multi-omics integration (mixOmics, MOFA)

  • Create multi-level regulatory networks

• Validation experiments:

  • Test predicted regulatory relationships using reporter assays

  • Perform perturbation experiments (overexpression, knockout) to verify network connections

  • Use time-course experiments to establish cause-effect relationships
    This integrated approach will provide a comprehensive understanding of ERF118 function within the broader context of plant development and stress responses, revealing both transcriptional and post-transcriptional regulatory mechanisms .

How can CRISPR-based approaches be combined with ERF118 antibodies for functional studies?

Integrating CRISPR technology with ERF118 antibody applications:

• Gene tagging strategies:

  • Use CRISPR to insert epitope tags (FLAG, HA) at the endogenous ERF118 locus

  • Create fluorescent protein fusions at endogenous loci for live imaging

  • Develop CRISPR activation/inhibition systems targeting ERF118 promoter

• Functional domain analysis:

  • Generate precise domain deletions or mutations

  • Create chimeric proteins with domains from related ERFs

  • Introduce phosphomimetic or phosphonull mutations at specific sites

  • Validate functional consequences using ERF118 antibodies

• Regulatory network mapping:

  • Perform CRISPR screens to identify genes affecting ERF118 expression/localization

  • Use antibodies to assess ERF118 levels/modification in CRISPR-modified backgrounds

  • Create reporter systems linked to ERF118 binding sites

• Technical considerations:

  • Validate CRISPR edits using antibody-based methods (Western blot, immunofluorescence)

  • Confirm maintained epitope recognition in modified proteins

  • Use CRISPR-modified lines as specificity controls for antibodies
    This integrated approach combines the precision of CRISPR gene editing with the detection capabilities of antibodies to provide comprehensive insights into ERF118 function and regulation .

What are the prospects for using ERF118 antibodies in single-cell protein analysis of plant tissues?

Single-cell protein analysis with ERF118 antibodies:

• Technical approaches:

  • Adapt flow cytometry protocols for plant protoplasts

  • Develop antibody-based CyTOF (mass cytometry) for plant cells

  • Apply immunofluorescence on tissue sections with single-cell resolution

  • Consider microfluidic approaches for protein analysis in isolated cells

• Method optimization:

  • Refine protoplast isolation protocols to maintain protein integrity

  • Develop fixation methods compatible with single-cell analysis

  • Optimize antibody penetration for intact tissue imaging

  • Create multiplexed antibody panels for simultaneous detection of ERF118 and related proteins

• Data analysis:

  • Apply single-cell clustering algorithms to identify cell populations

  • Correlate ERF118 expression with cell type markers

  • Perform pseudotime analysis to track developmental trajectories

  • Integrate with single-cell transcriptomics data

• Applications:

  • Map cell-specific responses to ethylene or stress treatments

  • Identify rare cell populations with unique ERF118 expression patterns

  • Analyze heterogeneity in meristematic tissues

  • Track dynamic changes during developmental processes
    This emerging approach would reveal cell-type specific regulation of ERF118, providing unprecedented resolution of its role in plant development and stress responses.

How might ERF118 antibodies contribute to understanding plant adaptation to climate change stresses?

ERF118 antibodies in climate change research:

• Stress response studies:

  • Analyze ERF118 protein levels under extreme temperature, drought, flooding

  • Compare ERF118 phosphorylation status between resistant and sensitive varieties

  • Investigate subcellular localization changes during stress adaptation

  • Study protein-protein interactions specific to stress conditions

• Comparative analysis across species:

  • Develop cross-reactive ERF118 antibodies for multiple crop species

  • Compare post-translational modifications across climate-adapted varieties

  • Analyze conservation of ERF118 regulatory mechanisms in stress-tolerant relatives

• Field-to-lab approaches:

  • Collect samples from plants grown in natural stress environments

  • Preserve protein modifications using optimized field sampling protocols

  • Correlate ERF118 levels/modifications with stress tolerance phenotypes

  • Develop simplified immunoassays for field research

• Applications for crop improvement:

  • Screen germplasm collections for favorable ERF118 expression patterns

  • Monitor ERF118 in breeding populations to track stress adaptation alleles

  • Assess the impact of genetic modifications on ERF118 pathways

  • Study epigenetic effects on ERF118 expression across generations
    This research direction would connect molecular mechanisms to real-world climate adaptation, potentially informing the development of more resilient crops through targeted breeding or genetic engineering approaches .

How do ERF118 protein sequences and epitopes compare across different plant species?

ERF118 comparison across plant species:

What methodological adaptations are needed when using ERF118 antibodies across different plant families?

Adapting methods for cross-species applications:

• Antibody selection considerations:

  • Choose antibodies targeting highly conserved epitopes for cross-species applications

  • Validate antibodies using recombinant proteins from target species

  • Consider generating new antibodies against species-specific sequences when needed

• Protocol modifications for different plant families:

  • Adjust protein extraction buffers based on tissue type (more detergent for woody tissues)

  • Modify fixation times for immunohistochemistry based on tissue permeability

  • Optimize antigen retrieval conditions for different tissue types

  • Adjust antibody concentrations and incubation times for each species

• Cross-reactivity validation:

  • Use Western blotting to confirm expected molecular weight in new species

  • Include positive controls from well-characterized species

  • Perform peptide competition assays to confirm specificity

  • Consider knockout/knockdown validation in model species when possible

• Data interpretation:

  • Account for potential paralogs in polyploid species

  • Consider evolutionary distance when interpreting cross-reactivity patterns

  • Be aware of species-specific post-translational modifications that may affect recognition
    These adaptations will help ensure reliable results when extending ERF118 antibody applications beyond model species to crops or wild relatives .

How can new antibody engineering technologies improve ERF118 detection specificity and sensitivity?

Enhancing ERF118 antibody technology:

• Next-generation antibody formats:

  • Develop single-chain variable fragments (scFvs) for improved tissue penetration

  • Create bispecific antibodies targeting ERF118 and interacting partners

  • Engineer nanobodies for super-resolution microscopy applications

  • Develop aptamer-based alternatives for challenging applications

• Specificity enhancements:

  • Apply phage display selection against multiple ERF family members to eliminate cross-reactivity

  • Implement negative selection strategies against closely related ERFs

  • Use computational epitope prediction to target unique regions

  • Develop antibodies specific to post-translationally modified forms of ERF118

• Sensitivity improvements:

  • Engineer high-affinity variants through directed evolution

  • Develop signal amplification methods compatible with plant tissues

  • Create proximity-based detection systems (proximity ligation, FRET-based)

  • Implement multiplexed detection systems for simultaneous analysis of multiple ERFs

• Novel applications:

  • Develop intrabodies for in vivo tracking and perturbation

  • Create optogenetic-antibody fusions for light-controlled ERF118 degradation

  • Design antibody-enzyme fusions for localized catalytic activity
    These advanced approaches would overcome current limitations in specificity and sensitivity, enabling more precise studies of ERF118 in complex plant systems .

What are the prospects for integrating ERF118 antibody approaches with emerging plant single-cell genomics techniques?

Integration with single-cell genomics:

• Methodological integration:

  • Develop protocols for sequential protein (antibody-based) and RNA analysis from the same cells

  • Adapt CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) for plant systems

  • Implement spatial transcriptomics with antibody-based protein detection

  • Create workflow for correlating single-cell proteomics with transcriptomics

• Technical considerations:

  • Optimize cell isolation protocols to preserve both RNA and protein integrity

  • Develop fixation methods compatible with both antibody binding and RNA isolation

  • Create barcoding strategies for tracking cells through multi-omic workflows

  • Implement computational methods for integrating protein and RNA datasets

• Applications:

  • Map cell-type specific ERF118 protein levels and corresponding transcriptional networks

  • Identify discrepancies between mRNA and protein levels at single-cell resolution

  • Analyze protein-RNA correlations during developmental transitions

  • Investigate cell-specific responses to environmental signals

• Challenges and solutions:

  • Address plant cell wall barriers through optimized protoplasting or permeabilization

  • Develop methods for subcellular protein localization in conjunction with transcriptomics

  • Create computational frameworks for multi-modal data integration

  • Implement benchmarking standards for method validation
    This integrated approach would provide unprecedented insights into the relationship between ERF118 protein dynamics and transcriptional regulation at single-cell resolution .

How might ERF118 antibodies contribute to agricultural biotechnology and crop improvement strategies?

ERF118 antibodies in agricultural applications:

• Crop improvement applications:

  • Screen germplasm collections for ERF118 protein variants associated with stress tolerance

  • Monitor protein levels during breeding programs to track favorable alleles

  • Assess ERF118 modifications in response to field conditions

  • Evaluate transgenic/gene-edited crops for desired ERF118 expression patterns

• Physiological monitoring:

  • Develop field-deployable immunoassays for ERF118 as stress response indicators

  • Create biosensor systems for real-time monitoring of ERF118 in growing plants

  • Implement high-throughput screening platforms for ERF118 responses to environmental factors

  • Design tissue-specific reporter systems based on ERF118 binding sites

• Integration with precision agriculture:

  • Correlate ERF118 protein patterns with remote sensing data

  • Develop predictive models connecting molecular markers to field performance

  • Create decision support tools using ERF118 status as input parameters

  • Implement targeted intervention strategies based on molecular diagnostics

• Enhanced breeding strategies:

  • Use ERF118 protein profiles as selection criteria in speed breeding programs

  • Develop ERF118-based markers for marker-assisted selection

  • Characterize germplasm resources based on ERF118 variants and modifications

  • Implement genome editing strategies targeting ERF118 regulatory networks
    These applications would translate fundamental knowledge about ERF118 into practical tools for crop improvement, particularly for enhancing stress resilience in changing climates .