ETO1 Antibody

What is ETO1 and what are the key applications for ETO1 antibodies in plant research?

ETO1 (ETHYLENE-OVERPRODUCER1) is a negative regulator of ethylene biosynthesis in plants, particularly Arabidopsis thaliana. It functions by directly inhibiting the enzymatic activity of type 2 ACC synthases (ACS), which are rate-limiting enzymes in the ethylene biosynthetic pathway. Additionally, ETO1 promotes the degradation of these ACS enzymes through the ubiquitin-proteasome pathway by interacting with CUL3, a component of E3 ubiquitin ligase complexes .
Key applications for ETO1 antibodies include:

• Western blotting to detect ETO1 protein levels and assess protein stability

• Immunoprecipitation to identify protein interaction partners

• Immunohistochemistry to localize ETO1 in plant tissues

• Studying the dynamics of ETO1-ACS-CUL3 complex formation

• Investigating the regulatory mechanisms of ethylene biosynthesis
  It's important to note that there is another protein abbreviated as ETO in human research (RUNX1T1/ETO, also known as Eight Twenty One protein), which is involved in acute myelogenous leukemia. Researchers should be careful to distinguish between these two unrelated proteins that share similar abbreviations .

What structural domains are present in ETO1 protein and how do they affect antibody selection?

The ETO1 protein contains several key structural domains that should be considered when selecting antibodies:

• BTB (Broad complex, Tramtrack, Bric-a-brac) domain - Essential for:

  • Homodimerization with other ETO1 proteins

  • Heterodimerization with EOL (ETO1-LIKE) proteins

  • Interaction with CUL3 (CULLIN3)

• TPR (Tetratricopeptide repeat) motifs - Critical for:

  • Interaction with target proteins, particularly type 2 ACS enzymes

  • Maintaining the proper structural configuration for function

• Linker sequences between TPR motifs - These are not merely spacers but play essential roles in the functionality of ETO1 .
  When selecting antibodies, researchers should consider:

• Epitope location relative to these functional domains

• Whether the antibody might interfere with protein-protein interactions

• If the epitope is accessible in native protein conformations

• Whether post-translational modifications might affect antibody recognition
  The BTB domain is sufficient for interaction with CUL3 and required for dimerization but isn't sufficient for the full spectrum of ETO1 function, indicating that antibodies targeting different domains may yield different results in functional studies .

How do researchers distinguish between ETO1 and EOL family proteins when designing experiments?

Distinguishing between ETO1, EOL1, and EOL2 proteins presents several methodological challenges that require careful experimental design:

• Antibody specificity:

  • Use antibodies raised against unique regions that differ between ETO1 and EOL proteins

  • Validate antibody specificity using knockout/knockdown lines for each protein

  • Consider epitope-tagged versions of these proteins when specific antibodies are unavailable

• Expression analysis approaches:

  • RT-qPCR with gene-specific primers to distinguish between transcripts

  • RNA-seq analysis with careful read mapping to discriminate between similar sequences

  • Reporter gene constructs with promoters from individual family members

• Functional analysis methods:

  • Utilize single, double, and triple mutants to assess functional redundancy

  • Complementation studies with specific family members under native promoters

  • Domain-swapping experiments between family members to identify functional differences

• Protein interaction studies:

  • Yeast two-hybrid assays with specific controls for each family member

  • Co-immunoprecipitation with tagged versions of each protein

  • In vitro binding assays with purified recombinant proteins
    ETO1, EOL1, and EOL2 show overlapping but distinct tissue-specific expression patterns, and neither EOL1 nor EOL2 can fully complement the eto1 phenotype when expressed under the control of the ETO1 promoter, suggesting unique functional properties of ETO1 .

What are the recommended protocols for using ETO1 antibodies in Western blotting?

Based on published research protocols, the following procedures are recommended for Western blotting with ETO1 antibodies:
Sample Preparation:

• Extract proteins from fresh plant tissue in buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, and protease inhibitor cocktail

• Quantify protein concentration using Bradford or BCA assay

• Mix samples with Laemmli buffer and heat at 95°C for 5 minutes
  Gel Electrophoresis:

• Load 50μg of protein sample per lane on a 5-20% SDS-PAGE gel

• Run at 70V (stacking gel) then 90V (resolving gel) for 2-3 hours
  Transfer:

• Transfer proteins to nitrocellulose membrane at 150mA for 50-90 minutes

• Verify transfer efficiency with Ponceau S staining
  Blocking and Antibody Incubation:

• Block membrane with 5% non-fat milk in TBS for 1.5 hours at room temperature

• Incubate with primary anti-ETO1 antibody (0.5-1.0 μg/mL) overnight at 4°C

• Wash with TBS-0.1% Tween three times for 5 minutes each

• Incubate with HRP-conjugated secondary antibody (1:10,000 dilution) for 1.5 hours at room temperature
  Detection:

• Develop using enhanced chemiluminescence (ECL) detection system

• Expected band size for ETO1 is approximately 70-75 kDa

• Expected band size for human RUNX1T1/ETO is approximately 67 kDa
  Including appropriate controls is essential: use wild-type tissue as a positive control and eto1 mutant tissue as a negative control to confirm antibody specificity.

What experimental techniques are commonly used to study ETO1 function using antibodies?

Several experimental techniques effectively utilize ETO1 antibodies to study protein function:

• Immunoprecipitation (IP):

  • Used to isolate ETO1 protein complexes from plant extracts

  • Helps identify interaction partners such as CUL3, ACS isozymes, and EOL proteins

  • Can be combined with mass spectrometry for unbiased identification of novel interactions

• Chromatin Immunoprecipitation (ChIP):

  • Although ETO1 is not a transcription factor, ChIP can be used to study its potential association with chromatin-bound protein complexes

  • Useful for investigating the regulatory mechanisms affecting ETO1 target genes

• Immunohistochemistry (IHC):

  • Localizes ETO1 expression in different plant tissues

  • Protocol example: Paraffin-embedded sections treated with heat-mediated antigen retrieval in citrate buffer (pH6) for 20 minutes, blocked with 10% goat serum, incubated with 1μg/ml anti-ETO1 antibody overnight at 4°C, detected using biotinylated secondary antibody and DAB chromogen

• Immunocytochemistry (ICC):

  • Determines subcellular localization of ETO1

  • Often combined with fluorescent markers for co-localization studies

• Flow Cytometry:

  • Quantifies ETO1 protein levels in protoplasts or isolated cells

  • Allows for high-throughput analysis of protein expression

• ELISA (Enzyme-Linked Immunosorbent Assay):

  • Provides quantitative measurement of ETO1 protein levels

  • Useful for comparing expression levels across different conditions or genotypes

• Proximity Ligation Assay (PLA):

  • Detects protein-protein interactions in situ

  • Especially valuable for studying the ETO1-ACS-CUL3 complex formation in intact cells
    These techniques provide complementary information about ETO1 function, localization, and protein interactions when used with specific, validated antibodies .

How does the BTB domain of ETO1 contribute to protein-protein interactions and what methodologies best capture these dynamics?

The BTB domain of ETO1 plays critical roles in multiple protein-protein interactions that are essential for regulating ethylene biosynthesis. Research findings indicate:

• Structural Role in Complex Formation:

  • The BTB domain is both necessary and sufficient for interaction with CUL3

  • It is required for homodimerization with other ETO1 proteins and heterodimerization with EOL1/2

  • The F466I substitution in eto1-5 significantly impairs dimerization without affecting binding to CUL3 or ACS5

• Methodological Approaches to Study BTB-Mediated Interactions:

  Method	Application	Technical Considerations
  Yeast Two-Hybrid	Initial screening of interaction partners	Use truncated constructs to map interaction domains
  Pull-down Assays	In vitro confirmation of direct interactions	Optimize buffer conditions (salt, pH) to maintain native interactions
  BiFC	Visualizing interactions in planta	Control for potential self-assembly of fluorescent protein fragments
  FRET/FLIM	Measuring interaction dynamics in live cells	Requires careful controls for fluorophore behavior
  Co-IP	Isolating native protein complexes	Use mild detergents and physiological salt concentrations
  SEC-MALS	Determining complex stoichiometry	Requires highly purified proteins

• Functional Significance:

  • Overexpression of ETO1 F466I in Arabidopsis results in a constitutive triple response phenotype in dark-grown seedlings

  • This demonstrates that dimerization is essential for proper regulation of ethylene biosynthesis
    When designing experiments to study BTB domain interactions, researchers should consider using domain-swapping approaches between ETO1 and EOL proteins, as well as site-directed mutagenesis of key residues to create separation-of-function mutants that specifically disrupt particular interactions while preserving others .

What is the mechanistic basis for ETO1's specificity toward type 2 ACS isozymes and how can this be investigated with antibodies?

ETO1 exhibits remarkable specificity for type 2 ACS isozymes through specific structural recognition mechanisms that can be investigated using antibody-based approaches:

• Molecular Basis of Specificity:

  • Yeast two-hybrid analysis using chimeric constructs between type 1 (LE-ACS2) and type 2 (LE-ACS3) ACS proteins revealed that the type-2-ACS-specific C-terminal tail is required for interaction with ETO1

  • Type 2 ACS isozymes (like AtACS5 and LE-ACS3) possess a characteristic C-terminal consensus sequence that is absent in type 1 (like LE-ACS2) and type 3 (like LE-ACS4) isozymes

  • The TPR motifs in ETO1 likely recognize and bind this specific C-terminal sequence

• Antibody-Based Approaches to Study Specificity:
  a. Co-immunoprecipitation with Domain-Specific Antibodies:

  • Use antibodies targeting different domains of ETO1 to map the regions involved in ACS recognition

  • Perform co-IP followed by western blot detection of specific ACS isozymes

  • Compare interaction patterns between wild-type and mutant proteins
    b. Competitive Binding Assays:

  • Utilize antibodies against the C-terminal region of type 2 ACS to block interaction with ETO1

  • Measure how this affects complex formation and ACS stability

  • Compare with control antibodies targeting other regions
    c. In situ Proximity Ligation Assay (PLA):

  • Use antibodies against ETO1 and different ACS isozymes

  • Visualize specific interactions in plant cells

  • Quantify interaction signals across different cell types and conditions

• Experimental Evidence:

  • When tomato was transformed with Arabidopsis ETO1, it interacted with tomato type 2 ACS (LE-ACS3) but not with LE-ACS2 (type 1) or LE-ACS4 (type 3)

  • Seedlings overexpressing ETO1 produced less ethylene than wild type when treated with auxin to induce LE-ACS3, despite comparable gene expression levels
    For researchers investigating this specificity, directed mutagenesis of the C-terminal region of type 2 ACS proteins combined with antibody-based detection methods can help identify the exact residues critical for ETO1 recognition and binding.

How can researchers optimize immunoprecipitation conditions for studying dynamic ETO1 complexes?

Optimizing immunoprecipitation (IP) conditions is critical for successfully capturing and studying the dynamic ETO1 protein complexes:

• Buffer Optimization for Complex Stability:

  Component	Recommended Range	Rationale
  Detergent	0.1-0.5% NP-40 or Triton X-100	Mild detergents preserve interactions
  Salt	100-150mM NaCl	Physiological concentration maintains specific interactions
  pH	7.2-7.5	Optimal for most plant protein interactions
  Protease inhibitors	Complete cocktail	Prevents degradation during extraction
  Phosphatase inhibitors	1mM NaF, 1mM Na₃VO₄	Preserves phosphorylation states if relevant
  Proteasome inhibitor	50μM MG132	Stabilizes ACS proteins targeted for degradation

• Antibody Selection Considerations:

  • Use affinity-purified antibodies specific to ETO1

  • Consider antibodies targeting different epitopes to avoid interference with interaction sites

  • For co-IP of ETO1 with ACS proteins, antibodies against the N-terminal region of ETO1 may be preferable to avoid interfering with C-terminal TPR domains that interact with ACS

  • Pre-clearing lysates with protein A/G beads reduces non-specific binding

• Crosslinking Strategies:

  • For transient or weak interactions, consider in vivo crosslinking with 1% formaldehyde for 10 minutes

  • DSP (dithiobis(succinimidyl propionate)) provides a reversible crosslink for complex stabilization

  • Optimize crosslinking time carefully to avoid over-fixation

• Technical Protocol Refinements:

  • Use fresh plant material and maintain cold temperatures throughout extraction

  • Include 5-10% glycerol in buffers to stabilize protein complexes

  • Consider sequential immunoprecipitation to isolate specific subcomplexes

  • For ubiquitination studies, include deubiquitinase inhibitors (N-ethylmaleimide)

• Controls and Validation:

  • Include IgG control IP to identify non-specific binding proteins

  • Use eto1 mutant tissue as a negative control

  • Confirm results with reciprocal IP (pull down with antibody against interaction partner)

  • Validate novel interactions with orthogonal methods (yeast two-hybrid, in vitro binding)
    By carefully optimizing these conditions, researchers can effectively capture and study the dynamic protein complexes involving ETO1, ACS, CUL3, and EOL proteins to better understand their roles in regulating ethylene biosynthesis.

What experimental strategies can resolve the distinct functions of ETO1 and EOL proteins in vivo?

Distinguishing between the functions of the structurally similar ETO1 and EOL proteins requires sophisticated experimental approaches that can detect subtle functional differences:

• Domain-Specific Antibody Generation:

  • Develop antibodies against unique regions that differ between family members

  • Use peptide competition assays to confirm specificity

  • Validate antibodies in single, double, and triple mutant backgrounds

• Combined Genetic and Biochemical Approaches:

  Approach	Methodology	Outcome Measure
  CRISPR/Cas9 editing	Create precise mutations in specific domains	Phenotypic analysis, protein interaction profiles
  Domain swapping	Generate chimeric proteins between family members	Complementation efficiency, protein stability
  Tissue-specific expression	Use promoters with distinct expression patterns	Spatial rescue of mutant phenotypes
  Inducible systems	Control protein expression temporally	Dynamic response to ethylene signals
  Epitope tagging	Tag each family member differently	Differential purification and detection

• Quantitative Proteomics:

  • Immunoprecipitate each family member separately

  • Use mass spectrometry to identify and quantify interaction partners

  • Apply SILAC or TMT labeling for comparative analysis

  • Analyze post-translational modifications specific to each protein

• High-Resolution Imaging:

  • Use super-resolution microscopy with specific antibodies

  • Track protein dynamics with photoactivatable fluorescent fusion proteins

  • Apply FRAP (Fluorescence Recovery After Photobleaching) to measure protein mobility

• Functional Readouts:

  • Develop in vitro assays measuring the direct effect on ACS activity

  • Quantify ACS protein stability in the presence of different family members

  • Measure ethylene production in various genetic backgrounds under controlled conditions

  • Analyze seedling responses to ethylene in single, double, and triple mutants
    By combining these approaches with carefully validated antibodies, researchers can better understand the distinct and overlapping functions of ETO1 and EOL proteins in regulating ethylene biosynthesis .

How do post-translational modifications of ETO1 affect its function and how can they be detected with antibodies?

Post-translational modifications (PTMs) of ETO1 play critical roles in regulating its activity, stability, and interactions. Detecting and characterizing these modifications requires specialized antibody-based approaches:

• Key Post-Translational Modifications of ETO1:

  • Phosphorylation: Likely regulates protein-protein interactions and complex formation

  • Ubiquitination: May control ETO1 stability and turnover

  • SUMOylation: Could affect protein localization and function

  • Redox modifications: May respond to stress conditions affecting ethylene biosynthesis

• Antibody-Based Detection Strategies:

  Modification	Detection Method	Technical Considerations
  Phosphorylation	Phospho-specific antibodies	Develop antibodies against predicted phosphorylation sites
  	Phosphatase treatment	Compare western blots before/after treatment
  	Phos-tag™ gels	Resolve phosphorylated forms without specific antibodies
  Ubiquitination	Anti-ubiquitin antibodies	Use after ETO1 immunoprecipitation
  	Tandem ubiquitin binding entities (TUBEs)	Enrich ubiquitinated proteins before detection
  SUMOylation	Anti-SUMO antibodies	IP under denaturing conditions to preserve modification
  Redox changes	Redox-sensitive probes	Capture transient modifications during stress

• Mass Spectrometry Integration:

  • Immunoprecipitate ETO1 using specific antibodies

  • Analyze by LC-MS/MS to map modification sites

  • Use SILAC or similar approaches to quantify modification changes

  • Compare modifications across different conditions or treatments

• Functional Studies of Modified ETO1:

  • Generate phospho-mimetic or phospho-dead mutations at identified sites

  • Test the impact on protein-protein interactions and ethylene regulation

  • Use site-specific antibodies to monitor modification status during development or stress

  • Correlate modification patterns with functional outcomes

• Methodological Considerations:

  • Include appropriate inhibitors during extraction (phosphatase inhibitors, deubiquitinase inhibitors)

  • Use rapid, gentle extraction methods to preserve labile modifications

  • Consider cell-free systems to study modification dynamics in vitro

  • Validate with genetic approaches (e.g., kinase mutants)
    Understanding how PTMs regulate ETO1 function could provide insights into the fine-tuning of ethylene biosynthesis regulation and potential targets for modulating ethylene production in plants under different conditions.

What is the optimal protocol for immunohistochemistry detection of ETO1 in plant tissues?

Based on published methods, the following optimized protocol is recommended for immunohistochemical detection of ETO1 in plant tissues:
Materials Required:

• Anti-ETO1 primary antibody

• Biotinylated secondary antibody

• Streptavidin-Biotin-Complex (SABC)

• DAB (3,3'-diaminobenzidine) substrate

• Citrate buffer (pH 6.0)

• Goat serum (10%)

• Plant tissue fixative (4% paraformaldehyde)

• Paraffin embedding materials

• Standard histology equipment
  Protocol:

• Tissue Preparation:

  • Fix plant tissues in 4% paraformaldehyde for 24 hours at 4°C

  • Dehydrate through ethanol series (30%, 50%, 70%, 85%, 95%, 100%)

  • Clear with xylene and embed in paraffin

  • Section at 5-10 μm thickness and mount on adhesive slides

  • Deparaffinize and rehydrate sections before staining

• Antigen Retrieval:

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

  • Heat for 20 minutes in a pressure cooker or microwave

  • Allow sections to cool for 20 minutes at room temperature

• Blocking and Antibody Incubation:

  • Block with 10% goat serum for 30-60 minutes at room temperature

  • Incubate with primary anti-ETO1 antibody (1μg/ml) overnight at 4°C

  • Wash 3 times with PBS, 5 minutes each

  • Incubate with biotinylated goat anti-rabbit IgG for 30 minutes at 37°C

  • Wash 3 times with PBS, 5 minutes each

• Detection:

  • Apply Streptavidin-Biotin-Complex (SABC) for 30 minutes at 37°C

  • Wash 3 times with PBS, 5 minutes each

  • Develop with DAB until desired staining intensity (3-10 minutes)

  • Counterstain with hematoxylin if desired

  • Dehydrate, clear, and mount with permanent mounting medium

• Controls:

  • Negative control: Omit primary antibody

  • Specificity control: Use eto1 mutant tissue

  • Positive control: Use tissue known to express ETO1 (based on transcriptional data)
    Optimization Tips:

• Carefully monitor antigen retrieval time as over-retrieval may damage tissue morphology

• Adjust primary antibody concentration based on expression levels in different tissues

• For fluorescent detection, replace biotinylated secondary with fluorophore-conjugated antibody

• For dual labeling, use antibodies raised in different host species and appropriate secondary antibodies
  This protocol has been successfully used to detect ETO1/RUNX1T1 in various tissue types and can be adjusted based on specific antibody characteristics and tissue properties .

How should researchers approach designing experiments to study the CUL3-ETO1-ACS protein complex?

Studying the CUL3-ETO1-ACS protein complex requires a multi-faceted experimental design that combines various techniques to capture this dynamic regulatory complex:

• Experimental Design Framework:

  Phase	Approach	Purpose
  Complex Verification	Co-immunoprecipitation with antibodies against each component	Confirm complex existence in vivo
  Interaction Mapping	Domain deletion and point mutations	Identify critical residues for interactions
  Functional Analysis	In vitro reconstitution	Assess complex activity under controlled conditions
  Dynamics Study	Time-course experiments	Track complex assembly/disassembly
  Structural Characterization	Crosslinking coupled with mass spectrometry	Map interaction surfaces

• Antibody-Based Methods:

  • Sequential immunoprecipitation: First IP with anti-ETO1, then elute and re-IP with anti-CUL3

  • Proximity-dependent biotin labeling (BioID): Fuse BioID to one component and identify proximal proteins

  • ChIP-like approaches to capture chromatin-associated complexes if relevant

  • Immunofluorescence co-localization to visualize complex formation in situ

• Biochemical Approaches:

  • Express and purify recombinant components (CUL3, ETO1, ACS)

  • Reconstitute complex in vitro under controlled conditions

  • Perform in vitro ubiquitination assays to assess functional activity

  • Use size exclusion chromatography to analyze complex stoichiometry

• Genetic Strategies:

  • Create separation-of-function mutants that disrupt specific interactions

  • Use inducible expression systems to control complex formation

  • Apply CRISPR/Cas9 to introduce specific mutations in endogenous genes

  • Generate fluorescent protein fusions for live imaging

• Critical Controls and Considerations:

  • Include proteasome inhibitors (MG132) to stabilize ubiquitinated intermediates

  • Compare complex formation under ethylene-inducing and non-inducing conditions

  • Consider tissue specificity and developmental timing

  • Use multiple antibodies targeting different epitopes to validate results

• Advanced Techniques:

  • Cryo-electron microscopy for structural analysis of the entire complex

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Single-molecule approaches to study complex dynamics

  • Computational modeling to predict complex behavior and generate testable hypotheses
    By systematically applying these approaches with well-validated antibodies, researchers can gain comprehensive insights into how the CUL3-ETO1-ACS complex assembles, functions, and regulates ethylene biosynthesis in plants .

What quality control measures should be implemented when using ETO1 antibodies?

Implementing robust quality control measures is essential when using ETO1 antibodies to ensure reliable and reproducible results:

• Antibody Validation Pipeline:

  Validation Step	Method	Acceptance Criteria
  Specificity Testing	Western blot	Single band of expected size (70-75 kDa for plant ETO1)
  	Immunoprecipitation followed by mass spectrometry	ETO1 identified as major hit
  	Knockout/knockdown controls	Significant reduction or absence of signal
  Cross-reactivity Assessment	Protein array testing	No significant binding to unrelated proteins
  	Testing against EOL1/EOL2	Quantifiable specificity for ETO1 vs. related proteins
  Lot-to-Lot Consistency	Comparison Western blots	Consistent band pattern and intensity
  	Standard sample testing	Consistent results between antibody lots

• Application-Specific Controls:

  • Western Blotting:

    • Include wild-type and eto1 mutant samples

    • Use recombinant ETO1 protein as positive control

    • Include molecular weight markers

    • Verify equal loading with appropriate controls (e.g., actin, tubulin)

  • Immunohistochemistry/Immunofluorescence:

    • Include no-primary-antibody control

    • Use competing peptide to confirm specificity

    • Include known expression pattern controls

    • Compare with mRNA expression patterns (in situ hybridization)

  • Immunoprecipitation:

    • Include IgG control IP

    • Verify pulled-down proteins by Western blot or mass spectrometry

    • Perform reciprocal IP with antibodies against interaction partners

• Antibody Storage and Handling:

  • Store according to manufacturer recommendations (typically at -20°C or -80°C)

  • Prepare single-use aliquots to avoid freeze-thaw cycles

  • Include carrier protein (BSA) for dilute antibody solutions

  • Monitor performance over time and with repeated use

• Documentation and Reporting Standards:

  • Record complete antibody information (source, catalog number, lot number)

  • Document all validation experiments performed

  • Include detailed methods sections in publications

  • Consider depositing validation data in public antibody validation repositories

• Advanced Validation Approaches:

  • Generate epitope-tagged ETO1 for parallel detection

  • Use multiple antibodies targeting different epitopes

  • Apply CRISPR/Cas9 to create epitope-modified endogenous ETO1

  • Validate key findings with orthogonal non-antibody methods
    Implementing these quality control measures will ensure that results obtained with ETO1 antibodies are specific, reproducible, and biologically meaningful, particularly when studying the complex regulatory mechanisms of ethylene biosynthesis .