ERF110 Antibody

What is ERF110 and why is it significant in plant biology research?

ERF110 is a transcription factor belonging to the B4 subfamily of the ERF (Ethylene Response Factor) protein family. It plays a crucial role in ethylene signaling pathways in plants, particularly Arabidopsis thaliana. The significance of ERF110 lies in its involvement in regulating flowering time (bolting) through a phosphorylation-dependent mechanism. Research has shown that ERF110 is phosphorylated at the Ser-62 position, and this specific phosphorylated isoform is required for normal Arabidopsis bolting by upregulating the flowering homeotic gene APETALA1 (AP1) at the transcriptional level .

Interestingly, ethylene has a dual effect on ERF110: it upregulates ERF110 gene expression via EIN2 while simultaneously downregulating its Ser-62 phosphorylation in an EIN2-independent manner. This dual regulation explains why both ethylene-response mutants and ERF110-deficient knockout lines share similar delayed-bolting phenotypes .

What types of ERF110 antibodies are currently available for research?

While specific commercial ERF110 antibody information is limited in the search results, researchers typically have access to several types of antibodies for plant transcription factors:

• Polyclonal antibodies: Generated against multiple epitopes of ERF110

• Monoclonal antibodies: Target specific ERF110 epitopes

• Phospho-specific antibodies: Specifically detect the Ser-62 phosphorylated form of ERF110

• Recombinant antibodies: Engineered for improved specificity

For ERF110 research, phospho-specific antibodies are particularly valuable as they can distinguish between the phosphorylated and non-phosphorylated forms of ERF110, enabling researchers to study the protein's functional state. Based on the methodology described in the literature, monoclonal antibodies raised against the Ser-62 phosphosite of ERF110 have been used to investigate ethylene-regulated phosphorylation .

How can I validate the specificity of an ERF110 antibody?

Validating antibody specificity is crucial for reliable research outcomes. For ERF110 antibodies, consider the following comprehensive validation approach:

• Western blot analysis: Compare protein extracts from wild-type plants and ERF110 knockout mutants (erf110-1 and erf110-2). A specific antibody should show a band at approximately the predicted molecular weight (~38-40 kDa) in wild-type samples that is absent in knockout mutants .

• Phospho-specificity validation: When using phospho-specific antibodies (like those targeting Ser-62 phosphorylated ERF110), compare samples with and without phosphatase treatment. The signal should diminish after phosphatase treatment .

• CRISPRi knockdown approach: Similar to methods used for other proteins, utilize CRISPRi to knockdown ERF110 expression. A specific antibody should show significantly reduced signal in knockdown samples compared to controls .

• Multiple reactivity tests: Test the antibody against recombinant ERF110 protein and related ERF family members to ensure specificity within the ERF family .

• Immunohistochemistry with controls: Perform parallel staining using isotype control antibodies and pre-immune serum to confirm specificity in tissue sections .

What are the optimal conditions for using ERF110 antibodies in Western blot analysis?

For optimal Western blot analysis using ERF110 antibodies, follow these methodological guidelines:

• Sample preparation:

  • Extract total proteins from plant tissues using a denaturing buffer containing phosphatase inhibitors (crucial for preserving phosphorylated ERF110)

  • Use fresh tissue whenever possible or flash-freeze in liquid nitrogen

  • Include protease inhibitors to prevent degradation

• Electrophoresis conditions:

  • Use 10-12% SDS-PAGE gels

  • Load 20-50 μg of total protein per lane

  • Include molecular weight markers spanning 25-75 kDa range

• Transfer and blocking:

  • Transfer proteins to PVDF membranes (preferred over nitrocellulose for phosphoproteins)

  • Block with 5% BSA in TBST (not milk, which contains phosphatases)

  • Incubate overnight at 4°C for maximum sensitivity

• Antibody dilutions:

  • Primary antibody: Start with 0.2-0.5 μg/ml for mouse IgG antibodies

  • Secondary antibody: Typically 1:5000-1:10000 dilution of HRP-conjugated anti-mouse or anti-rabbit IgG

• Detection:

  • Enhanced chemiluminescence (ECL) systems work well for detecting ERF110

  • For phospho-specific detection, consider using more sensitive detection methods

• Controls:

  • Always include positive control (wild-type plant extract)

  • Negative control (erf110 knockout mutant extract)

  • Loading control (anti-actin or anti-tubulin antibody)

How can I use phospho-specific ERF110 antibodies to investigate ethylene signaling pathways?

Phospho-specific ERF110 antibodies are powerful tools for dissecting ethylene signaling pathways in plants. Here's a comprehensive methodological approach:

• Temporal phosphorylation dynamics:

  • Treat plants with ethylene (or ethylene precursor ACC) at various time points

  • Prepare protein extracts with phosphatase inhibitors

  • Perform Western blot analysis using both total ERF110 antibody and phospho-Ser-62 specific antibody

  • Quantify the ratio of phosphorylated to total ERF110 to determine phosphorylation dynamics

• Pathway dissection using mutants:

  • Compare ERF110 phosphorylation levels in wild-type plants versus ethylene signaling mutants (ein2-5, etr1-1, ctr1-1)

  • This approach helps determine which components of the ethylene signaling pathway affect ERF110 phosphorylation

  • Based on previous research, ethylene downregulates Ser-62 phosphorylation in an EIN2-independent manner

• Kinase identification:

  • Perform in vitro kinase assays using synthesized peptides containing the Ser-62 phosphosite

  • Test kinase extracts from different ethylene-response mutants to identify the responsible kinase

  • Use immunoprecipitation with ERF110 antibodies followed by mass spectrometry to identify interacting kinases

• Subcellular localization changes:

  • Use immunofluorescence with phospho-specific antibodies to track changes in localization of phosphorylated ERF110 after ethylene treatment

  • Complement with GFP-ERF110 fusion proteins to confirm results

• Downstream target analysis:

  • Perform ChIP using ERF110 antibodies before and after ethylene treatment

  • Identify changes in DNA binding patterns correlated with phosphorylation state

  • Compare with known targets like AP1 to establish regulatory mechanisms

Can ERF110 antibodies be used for immunoprecipitation to identify protein interaction partners?

Yes, ERF110 antibodies can be effectively used for immunoprecipitation (IP) to identify protein interaction partners. Here's a detailed methodological approach:

• Sample preparation:

  • Use plant tissue at appropriate developmental stages (e.g., pre-bolting for flowering studies)

  • Extract proteins under native conditions to preserve protein-protein interactions

  • Consider crosslinking with formaldehyde to stabilize transient interactions

• Immunoprecipitation protocol:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Incubate cleared lysates with ERF110 antibody (typically 2-5 μg antibody per mg protein)

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

  • Wash extensively to remove non-specific binders

  • Elute bound proteins for downstream analysis

• Controls:

  • Include IgG isotype control immunoprecipitation

  • Perform parallel IP from erf110 knockout plants

  • Consider using Ser-62 phospho-specific antibodies to compare interaction partners of phosphorylated versus non-phosphorylated ERF110

• Mass spectrometry analysis:

  • Analyze eluted proteins by LC-MS/MS

  • Use label-free quantification to compare with control samples

  • Filter proteins enriched at least 2-fold over controls

• Validation of interactions:

  • Confirm key interactions by co-immunoprecipitation in reverse direction

  • Perform yeast two-hybrid or bimolecular fluorescence complementation assays

  • Test interaction with known components of ethylene signaling pathway

This approach has been successfully used for other transcription factors and can be adapted for ERF110 to identify components of the signaling network that regulates its phosphorylation and activity .

Why might I observe multiple bands when using ERF110 antibodies in Western blot?

Multiple bands in Western blots using ERF110 antibodies can occur for several reasons. Here's a systematic approach to identify and resolve this issue:

• Post-translational modifications:

  • ERF110 is known to be phosphorylated at Ser-62, which can cause a mobility shift

  • Other potential modifications (ubiquitination, SUMOylation) may create additional bands

  • Solution: Compare with phosphatase-treated samples to identify phosphorylation-dependent bands

• Protein degradation:

  • ERF transcription factors can be subject to rapid turnover

  • Lower molecular weight bands may represent degradation products

  • Solution: Use fresh samples, add protease inhibitors, and keep samples cold throughout processing

• Alternative splicing:

  • ERF genes can have alternative splice variants

  • Solution: Compare band patterns with information about known splice variants from genomic databases

• Cross-reactivity:

  • Antibodies may cross-react with related ERF family members

  • Solution: Test antibody specificity against recombinant proteins of closely related ERFs

• Non-specific binding:

  • Secondary antibody might recognize endogenous plant immunoglobulins

  • Solution: Test secondary antibody alone and optimize blocking conditions

• Experimental validation:

  • Confirm the identity of bands using ERF110 knockout or knockdown plants

  • For phospho-specific antibodies, compare band patterns after ethylene treatment, which is known to reduce Ser-62 phosphorylation

  • Consider using lambda phosphatase treatment to collapse multiple bands due to different phosphorylation states

How can I optimize immunohistochemistry protocols for detecting ERF110 in plant tissues?

Optimizing immunohistochemistry (IHC) protocols for ERF110 detection in plant tissues requires careful attention to fixation, antigen retrieval, and signal detection. Here's a comprehensive approach:

• Tissue fixation and processing:

  • Fix tissues in 4% paraformaldehyde for 12-24 hours

  • Consider alternative fixatives if phospho-epitopes are important

  • Process tissues carefully to avoid excessive heat during paraffin embedding

  • Cut thin sections (5-7 μm) for optimal antibody penetration

• Antigen retrieval:

  • Heat-induced epitope retrieval: Citrate buffer (pH 6.0) at 95°C for 20 minutes

  • For phospho-specific antibodies: Try sodium EDTA buffer (pH 8.0)

  • Allow sections to cool slowly (20-30 minutes) to room temperature

• Blocking and antibody incubation:

  • Block with 5% BSA and 5% normal serum from secondary antibody host species

  • Add 0.1% Triton X-100 for membrane permeabilization

  • Use primary antibody concentration of 2-5 μg/ml for mouse IgG antibodies

  • Incubate overnight at 4°C in a humidified chamber

• Signal detection optimization:

  • Try both chromogenic (DAB) and fluorescent detection systems

  • For fluorescence, consider using tyramide signal amplification for low-abundance targets

  • Use DAPI or other nuclear counterstains to help visualize cellular structure

• Controls and validation:

  • Include negative control sections (no primary antibody)

  • Use tissues from erf110 knockout plants as specificity controls

  • Consider dual staining with markers for subcellular compartments to confirm localization

• Troubleshooting common issues:

  • High background: Increase blocking time, optimize antibody dilution

  • No signal: Try different antigen retrieval methods, increase antibody concentration

  • Non-specific signal: Pre-adsorb antibody with plant tissue extract from knockout plants

By systematically optimizing each step, you can develop a reliable IHC protocol for detecting ERF110 in plant tissues .

How can I generate and validate phospho-specific antibodies against ERF110 Ser-62?

Generating and validating phospho-specific antibodies against ERF110 Ser-62 requires careful planning and rigorous testing. Here's a comprehensive methodological approach:

• Peptide design and synthesis:

  • Design a phosphopeptide spanning 10-15 amino acids centered on Ser-62

  • Include the sequence context: VDpSSHNPIEESMSK (where pS represents phospho-Ser-62)

  • Synthesize both phosphorylated and non-phosphorylated versions of the peptide

  • Add a C-terminal cysteine for conjugation to carrier protein

• Immunization strategy:

  • Conjugate the phosphopeptide to KLH (keyhole limpet hemocyanin)

  • Immunize rabbits or mice with the phosphopeptide-KLH conjugate

  • Use multiple animals to increase chances of success

  • Consider immunizing with non-phosphopeptide as well to generate total ERF110 antibodies

• Antibody purification:

  • Collect serum after sufficient immunization period

  • Perform initial affinity purification using the phosphopeptide

  • Remove antibodies that recognize non-phosphorylated epitopes by passing through a column with the non-phosphorylated peptide

• Validation tests:

  • ELISA: Test reactivity against phospho vs. non-phospho peptides

  • Western blot: Compare reactivity using:

    • Recombinant ERF110 protein with and without in vitro phosphorylation

    • Plant extracts from wild-type vs. erf110 knockout lines

    • Extracts from plants treated with and without ethylene (which reduces Ser-62 phosphorylation)

    • Extracts before and after phosphatase treatment

  • Immunoprecipitation: Verify ability to capture phosphorylated ERF110

  • Mass spectrometry: Confirm that immunoprecipitated protein is phosphorylated at Ser-62

• Characterization of phospho-specificity:

  • Test cross-reactivity with related ERF family proteins

  • Determine minimum detectable amount of phosphorylated ERF110

  • Establish the ratio of reactivity (phospho vs. non-phospho form)

This approach has been successfully used to generate and validate phospho-specific antibodies for ERF110 Ser-62, as evidenced by their application in ethylene signaling research .

What are the latest methods for using ERF110 antibodies in single-cell proteomics research?

Single-cell proteomics is a cutting-edge field, and adapting ERF110 antibodies for this application requires innovative approaches. Here's a methodological framework:

• Mass cytometry (CyTOF) adaptation:

  • Conjugate ERF110 antibodies to rare earth metals

  • Optimize metal-labeling density to ensure sensitivity

  • Develop protocols for plant protoplast preparation compatible with CyTOF

  • Include markers for cell types and cellular states

  • This approach allows simultaneous measurement of ERF110 and other proteins in thousands of individual cells

• Single-cell Western blot techniques:

  • Adapt microfluidic single-cell Western blot platforms for plant protoplasts

  • Optimize lysis conditions to preserve phosphorylation states

  • Use fluorescently-labeled ERF110 antibodies alongside markers for cell types

  • Quantify ERF110 expression and phosphorylation at single-cell resolution

• Proximity ligation assay (PLA) for interaction studies:

  • Combine ERF110 antibodies with antibodies against potential interaction partners

  • Use PLA to visualize and quantify protein interactions in situ

  • Apply to tissue sections to maintain spatial context

  • This approach can reveal cell-type specific interactions of ERF110

• Spatial proteomics using multiplexed immunofluorescence:

  • Employ cyclic immunofluorescence with ERF110 antibodies

  • Strip and reprobe the same tissue section multiple times

  • Build comprehensive spatial maps of ERF110 in relation to other proteins

  • Integrate with single-cell transcriptomics data

• Next-generation antibody barcoding:

  • Adapt DNA-barcoded antibody techniques for ERF110 detection

  • Use oligonucleotide-conjugated ERF110 antibodies

  • Combine with single-cell sequencing platforms

  • This approach allows correlation of ERF110 protein levels with transcriptome profiles

• Validation strategies for single-cell applications:

  • Use CRISPR-engineered cell lines with tagged ERF110 as controls

  • Compare bulk measurements with aggregated single-cell data

  • Perform spike-in experiments with known quantities of recombinant protein

These cutting-edge approaches enable researchers to study ERF110 biology at unprecedented resolution, revealing cell-type specific regulation and heterogeneity in response to ethylene signaling.

How do monoclonal and polyclonal ERF110 antibodies compare in different experimental applications?

Monoclonal and polyclonal ERF110 antibodies each have distinct advantages and limitations depending on the experimental application. Here's a comparative analysis:

Methodological Recommendations:

• For detailed phosphorylation studies:

  • Use monoclonal phospho-specific antibodies targeting Ser-62

  • These provide precise detection of phosphorylation state changes after ethylene treatment

• For general ERF110 detection:

  • Polyclonal antibodies against full-length ERF110 are more versatile

  • Better for detecting ERF110 across different plant species due to epitope diversity

• For co-localization studies:

  • Monoclonal antibodies provide cleaner signals and less cross-reactivity

  • Important when using multiple antibodies simultaneously

• For evolutionarily conserved regions:

  • Generate antibodies against highly conserved domains for cross-species reactivity

  • Test using sequence alignment to predict cross-reactivity

• For quantitative analysis:

  • Monoclonal antibodies provide more consistent results for comparing ERF110 levels

  • Important for time-course experiments examining ethylene responses

The choice between monoclonal and polyclonal antibodies should be guided by the specific research question, required sensitivity, and experimental conditions .

What are the latest computational approaches for predicting optimal epitopes for ERF110 antibody generation?

Modern computational approaches have revolutionized epitope selection for antibody generation. For ERF110 antibodies, consider these cutting-edge methods:

• Structure-based epitope prediction:

  • Use AlphaFold2 or RoseTTAFold to predict the 3D structure of ERF110

  • Identify surface-exposed regions likely to be accessible to antibodies

  • Apply solvent accessibility calculations to rank potential epitopes

  • Focus on structurally stable regions that maintain conformation

• Machine learning-based epitope ranking:

  • Employ deep learning models trained on antibody-antigen complexes

  • Use models like those described in recent literature that incorporate physics- and AI-based methods

  • Score potential ERF110 epitopes based on:

    • Surface accessibility

    • Hydrophilicity

    • Sequence conservation

    • Secondary structure stability

• Developability-aware epitope selection:

  • Integrate developability predictions into epitope selection

  • Avoid regions prone to aggregation or poor solubility

  • Use computational tools to predict epitope stability and expression

  • Apply "Lab-in-the-loop" approaches combining computational prediction with experimental feedback

• Specificity optimization:

  • Compare ERF110 sequences with related ERF family proteins

  • Identify unique regions to minimize cross-reactivity

  • Use sequence alignment tools to highlight ERF110-specific regions

  • Apply specificity filters to exclude epitopes with similarity to other plant proteins

• Phospho-epitope optimization:

  • For phospho-specific antibodies, analyze the sequence context around Ser-62

  • Apply specialized phospho-epitope prediction algorithms

  • Consider the 3D environment of the phosphorylation site

  • Design epitopes that maximize exposure of the phosphorylated residue

• Experimental validation of predictions:

  • Synthesize peptides corresponding to top predicted epitopes

  • Test immunogenicity in silico and in vitro

  • Use phage display experiments for validation

  • Apply active learning approaches to refine predictions

These computational approaches significantly improve success rates in generating highly specific and effective ERF110 antibodies while reducing experimental iterations .

How might advanced genetic engineering approaches improve ERF110 antibody specificity and applications?

Advanced genetic engineering offers promising avenues to revolutionize ERF110 antibody development and applications:

• CRISPR-engineered epitope tagging:

  • Use CRISPR/Cas9 to insert small epitope tags into the endogenous ERF110 gene

  • Create knock-in plant lines with HA, FLAG, or HiBiT tags

  • Enable detection with highly specific commercial tag antibodies

  • Preserve native expression patterns and regulation

  • Generate phosphomimetic mutants (S62D) to study constitutively "active" forms

• Nanobody and single-domain antibody development:

  • Engineer camelid nanobodies against ERF110

  • Select for phospho-specific binding using phage display

  • Express intracellularly as "intrabodies" to track ERF110 in living cells

  • Fuse to fluorescent proteins for real-time imaging of ERF110 dynamics

• Split-protein complementation systems:

  • Create ERF110 fusion proteins with split GFP or luciferase fragments

  • Engineer complementary fragments fused to ERF110-interacting proteins

  • Visualize interactions in real-time during ethylene response

  • Apply to study dynamic phosphorylation-dependent interactions

• Genetically encoded biosensors:

  • Develop FRET-based sensors for ERF110 phosphorylation state

  • Design conformational sensors that respond to ERF110 activity

  • Create plant lines with stable integration of these sensors

  • Enable real-time tracking of ERF110 activity during development

• Antibody engineering for enhanced properties:

  • Apply computational design of developable therapeutic antibodies methods

  • Improve affinity, specificity, and stability of ERF110 antibodies

  • Employ phage display with next-generation sequencing to optimize binding

  • Use machine learning to predict successful antibody variants

• Multifunctional antibody conjugates:

  • Create ERF110 antibody-DNA conjugates for proximity labeling

  • Develop antibody-enzyme fusions for spatially-resolved proteomics

  • Engineer antibody-based degraders to control ERF110 protein levels

  • Design dual-specificity antibodies to study co-localization with other proteins

These advanced genetic engineering approaches could transform our ability to study ERF110 function and regulation in plant development and ethylene signaling .

What role might ERF110 antibodies play in understanding plant responses to climate change?

ERF110 antibodies could become essential tools for studying plant adaptation to climate change, offering unique insights into ethylene-mediated stress responses:

• Monitoring ERF110 phosphorylation under stress conditions:

  • Use phospho-specific antibodies to track Ser-62 phosphorylation states

  • Compare ERF110 activation patterns under:

    • Elevated temperatures

    • Drought conditions

    • Increased CO₂ concentrations

    • Combined stresses mimicking climate change scenarios

  • Establish correlations between phosphorylation patterns and adaptive responses

• Comparative studies across plant species:

  • Develop ERF110 antibodies that recognize conserved epitopes

  • Compare ERF110 regulation in:

    • Crop plants with different climate tolerances

    • Wild relatives adapted to extreme environments

    • Model species under controlled climate change simulations

  • Identify conserved and divergent regulatory mechanisms

• High-throughput screening applications:

  • Create ERF110 antibody-based assays for screening germplasm

  • Develop ELISA or protein microarray methods to quantify ERF110 activity

  • Screen large populations for variation in ERF110 regulation

  • Identify lines with enhanced adaptive capacity via altered ERF110 signaling

• Tissue-specific response mapping:

  • Use immunohistochemistry to map ERF110 activation patterns

  • Compare cellular responses across tissues under stress conditions

  • Identify key sites of ethylene-mediated adaptation

  • Correlate with developmental transitions affected by climate conditions

• Integration with multi-omics approaches:

  • Combine ERF110 antibody-based proteomics with:

    • Transcriptomics to correlate protein activity and gene expression

    • Metabolomics to link ERF110 activity with metabolic adaptations

    • Phenomics to connect molecular changes to whole-plant responses

  • Build predictive models of plant responses to climate variables

• Developmental timing studies:

  • Track ERF110 phosphorylation during key developmental transitions

  • Examine how climate stressors alter the timing of ERF110 activation

  • Study the impact on flowering time and reproductive success

  • Connect molecular mechanisms to population-level climate adaptation