EIN4 Antibody

What is EIN4 and why is it important for plant biology research?

EIN4 is a member of the five-member ethylene-receptor family in Arabidopsis thaliana, which includes ETR1, ETR2, EIN4, and ERS1. These receptors play crucial roles in ethylene signaling pathways that regulate numerous plant growth and developmental processes. The EIN4 receptor is particularly important because mutant analyses have shown that the etr1 etr2 ein4 triple mutant exhibits a partial constitutive ethylene-response phenotype, resulting in reduced shoot growth and shorter hypocotyls in air compared to wild-type plants . Understanding EIN4 function provides valuable insights into plant hormone signaling networks, stress responses, and developmental regulation. Antibodies against EIN4 are therefore essential tools for investigating these processes at the molecular level.

How do EIN4 antibodies differ from other ethylene receptor antibodies in terms of specificity and applications?

EIN4 antibodies are specifically designed to recognize epitopes unique to the EIN4 receptor, distinguishing it from other ethylene receptors like ETR1, ETR2, and ERS1. While all ethylene receptor antibodies can be used to study ethylene signaling, the choice of receptor-specific antibodies depends on the research question. ETR1 antibodies are often used for general ethylene receptor studies due to ETR1's predominant role, while EIN4 antibodies are particularly valuable when investigating receptor subfunctionalization or when the ein4 mutation shows unique phenotypes in combination with other receptor mutations . Researchers should carefully validate cross-reactivity between receptor family members through appropriate controls, as these receptors share conserved domains.

What are the common methodological approaches for EIN4 detection in plant tissues?

Several methodological approaches can be used for EIN4 detection:

• Immunoblotting/Western blot: Most commonly used to detect protein expression levels in tissue extracts. Typical protocols involve:

  • Tissue extraction with appropriate buffers containing protease inhibitors

  • Protein separation via SDS-PAGE (typically 8-10% gels)

  • Transfer to membrane and blocking

  • Primary incubation with EIN4 antibody (typically 1:1000-1:5000 dilution)

  • Secondary antibody application and detection

• Immunohistochemistry/Immunofluorescence: For localization studies within tissues and cells, similar to techniques used with other receptor antibodies .

• Immunoprecipitation: To study protein-protein interactions involving EIN4, following protocols similar to those used for other membrane receptors.

Each method requires optimization of antibody concentration, incubation conditions, and detection systems specific to plant tissues.

How should I design controls for EIN4 antibody specificity validation?

Proper validation of EIN4 antibody specificity requires several critical controls:

• Genetic controls: Include tissue samples from:

  • Wild-type plants (positive control)

  • ein4 knockout/null mutants (negative control)

  • Plants overexpressing EIN4 (enhanced signal control)

• Peptide competition assay: Pre-incubate the antibody with excess EIN4-specific peptide used for immunization to block specific binding sites before application to samples.

• Cross-reactivity assessment: Test the antibody against purified recombinant proteins or extracts from plants overexpressing other ethylene receptors (ETR1, ETR2, ERS1) to evaluate potential cross-reactivity.

• Secondary antibody-only control: Omit primary antibody to assess non-specific binding of secondary antibody.

These controls should be performed during initial antibody characterization and periodically during experimental use to ensure continued specificity .

What are optimal sample preparation methods for enhancing EIN4 detection in plant tissues?

Membrane-bound receptors like EIN4 require specialized extraction procedures:

• Buffer composition: Use buffers containing:

  • Mild detergents (0.5-1% Triton X-100 or NP-40)

  • Protease inhibitor cocktail

  • Reducing agents (DTT or β-mercaptoethanol)

  • Phosphatase inhibitors if studying phosphorylation status

• Tissue disruption:

  • For Arabidopsis, grind tissue in liquid nitrogen to fine powder

  • Maintain cold temperature throughout extraction

  • Consider ultrasonication for membrane protein solubilization

• Subcellular fractionation:

  • For enrichment of endoplasmic reticulum membranes where ethylene receptors localize

  • Use density gradient centrifugation methods

  • Verify fraction purity with appropriate markers

• Sample storage:

  • Prepare fresh samples when possible

  • If storage is necessary, flash-freeze in liquid nitrogen and store at -80°C

  • Avoid repeated freeze-thaw cycles

These methods increase detection sensitivity by preserving protein integrity and concentrating membrane-bound receptors .

How can immunoprecipitation be optimized for EIN4 interaction studies?

For successful EIN4 immunoprecipitation:

• Crosslinking considerations:

  • Chemical crosslinkers (DSP, formaldehyde) can stabilize transient interactions

  • Optimize crosslinking time (typically 10-30 minutes) to prevent over-crosslinking

  • Include uncrosslinked controls

• Antibody coupling:

  • Direct coupling to beads (e.g., NHS-activated sepharose) often improves results

  • Use spacer arms for better accessibility to epitopes

  • Determine optimal antibody:bead ratio (typically 5-10 μg antibody per 50 μl bead slurry)

• Washing conditions:

  • Balance stringency (salt concentration, detergent) against preservation of interactions

  • Include graduated washes of decreasing stringency

  • Perform control IPs with non-specific IgG to identify non-specific binding

• Elution strategies:

  • Competitive elution with epitope peptide for mild conditions

  • Low pH glycine buffers for stronger elution

  • SDS sample buffer for complete elution

• Verification:

  • Confirm successful IP by immunoblotting a fraction of the eluate

  • Analyze interacting partners by mass spectrometry

What strategies exist for differentiating between active and inactive conformations of EIN4 using antibodies?

Distinguishing between the "on" (ethylene-unbound) and "off" (ethylene-bound) conformations of ethylene receptors like EIN4 is challenging but possible using:

• Conformation-specific antibodies:

  • Generate antibodies against peptides representing known conformational states

  • Validate using receptors locked in specific conformations (e.g., through mutations)

  • Example: Antibodies targeting the D25 region of ETR1 might similarly be developed for EIN4's equivalent domain

• Proximity-based detection methods:

  • FRET or BRET-based approaches using fluorescent or bioluminescent tags

  • Bimolecular fluorescence complementation (BiFC) to visualize interaction-dependent conformational changes

  • These methods require genetic modification of the receptor

• Native versus denaturing conditions:

  • Some antibodies preferentially recognize native conformations

  • Compare antibody binding under native versus denaturing conditions

  • Use non-denaturing PAGE for conformation-dependent detection

• Crosslinking-based approaches:

  • Employ conformation-selective crosslinkers

  • Analyze crosslinked products by immunoblotting with EIN4 antibodies

These approaches require extensive optimization and validation but provide valuable insights into receptor activation states .

How can I resolve inconsistent EIN4 antibody staining patterns between experiments?

Inconsistent staining with EIN4 antibodies may result from several factors:

Additionally, include technical replicates and positive controls in each experiment. Consider creating a reference sample batch that can be run alongside experimental samples to calibrate between experiments .

What approaches exist for analyzing EIN4 receptor clustering and dimerization using antibody-based methods?

Several advanced techniques can be employed to study EIN4 receptor clustering and dimerization:

• Proximity ligation assay (PLA):

  • Uses pairs of antibodies against the same or different components

  • Provides visual signals when targets are within 40 nm

  • Can detect EIN4 homodimers or heterodimers with other ethylene receptors

  • Quantifiable by fluorescence microscopy

• Co-immunoprecipitation (Co-IP) with quantitative analysis:

  • Pull down with EIN4 antibody and probe for other receptors

  • Include crosslinking step to capture transient interactions

  • Use quantitative immunoblotting or mass spectrometry for measurement

  • Compare ratios under different conditions (e.g., with/without ethylene)

• Blue native PAGE:

  • Preserves protein complexes in their native state

  • Follow with immunoblotting using EIN4 antibodies

  • Analyze band sizes to determine complex formation

  • Compare patterns with/without treatments or in different mutant backgrounds

• Förster resonance energy transfer (FRET) with antibodies:

  • Use fluorescently labeled primary or secondary antibodies

  • Direct measurement of protein proximity in situ

  • Requires careful controls for non-specific binding

These methods provide complementary information about receptor interactions and should be used in combination for comprehensive analysis .

How should I quantify and normalize Western blot data for EIN4 expression studies?

Proper quantification and normalization of EIN4 Western blot data requires:

• Image acquisition:

  • Use a wide dynamic range imaging system (e.g., chemiluminescence imager with cooled CCD camera)

  • Ensure signals are within linear detection range (not saturated)

  • Take multiple exposures if necessary

• Quantification software:

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

  • Define lanes and bands consistently

  • Subtract local background for each band

• Normalization approaches:

  • Loading controls: Normalize to housekeeping proteins (e.g., actin, tubulin, GAPDH)

  • Total protein normalization: Use stain-free gels or total protein stains (SYPRO Ruby, Ponceau S)

  • Internal control: Include a reference sample on each gel for inter-gel normalization

• Statistical analysis:

  • Run at least three biological replicates

  • Perform appropriate statistical tests (t-test, ANOVA)

  • Report normalized data with error bars and significance values

• Validation:

  • Confirm key findings with alternative techniques (qRT-PCR, immunohistochemistry)

  • Check if protein levels correlate with known physiological responses

What criteria should be used to evaluate the quality and specificity of commercially available EIN4 antibodies?

When evaluating EIN4 antibodies, consider these critical criteria:

• Validation documentation:

  • Western blot images showing the expected band size (~77-80 kDa for EIN4)

  • Evidence of testing in both wild-type and ein4 mutant tissues

  • Cross-reactivity assessment with other ethylene receptors

• Antibody characteristics:

  • Clearly defined immunogen information (peptide sequence or protein domain)

  • Production method (monoclonal vs. polyclonal)

  • Host species and isotype information

  • Purification method

• Application-specific validation:

  • Tested in your specific application (WB, IHC, IP, etc.)

  • Optimized protocols for your application

  • Reported working dilutions and conditions

• Independent verification:

  • Published literature using the same antibody

  • Positive reviews from other researchers

  • Alternative antibodies targeting different epitopes for confirmation

• Lot-to-lot consistency:

  • Evidence of quality control between lots

  • Consistent performance across multiple batches

How can optical density (OD) measurements in EIN4 antibody-based assays be quantitatively interpreted?

Optical density measurements in antibody-based assays like ELISA require careful interpretation:

• Establishing threshold values:

  • Define positive/negative thresholds using:

    • ROC curve analysis with known positive/negative samples

    • Mean + 2-3 SD of negative control samples

    • Comparison to reference standards

• Quantitative interpretation of OD values:

  • Create standard curves using purified EIN4 protein at known concentrations

  • Analyze relationship between OD and antigen concentration:

    • Linear range typically 0.1-2.0 OD units

    • Sigmoidal relationship across wider ranges

  • Convert OD to concentration using four-parameter logistic regression

• Assessing result reliability:

  • Include internal controls on each plate

  • Calculate coefficient of variation (CV) between replicate wells

  • Consider results reliable when CV < 15%

• Understanding relationship to functional assays:

  • Higher OD values generally correlate with higher antibody concentrations

  • Example from other systems: In HIT antibody detection, OD values ≥1.40 units had ≥50% probability of platelet activation in functional assays

• Sources of variability to control:

  • Incubation timing and temperature

  • Washing efficiency

  • Substrate development time

  • Plate reader calibration and settings

This approach provides more meaningful interpretation beyond simple positive/negative designations .

How can EIN4 antibodies be utilized in studies of ethylene receptor crosstalk with other hormone signaling pathways?

EIN4 antibodies can provide valuable insights into hormone signaling network integration:

• Co-immunoprecipitation studies:

  • Use EIN4 antibodies to pull down receptor complexes

  • Analyze precipitates for components of other hormone signaling pathways

  • Mass spectrometry analysis can identify novel interactors

  • Western blotting for known components of auxin, cytokinin, or ABA pathways

• Proximity-based studies:

  • Proximity ligation assays (PLA) to visualize interactions in situ

  • BiFC with split fluorescent proteins to confirm direct interactions

  • FRET/FLIM using labeled antibodies to measure dynamic interactions

• Phosphorylation status analysis:

  • Immunoprecipitate EIN4 and probe with phospho-specific antibodies

  • Mass spectrometry to identify phosphorylation sites

  • Compare phosphorylation patterns after treatment with multiple hormones

• Receptor complex dynamics:

  • Track EIN4-containing complexes during simultaneous hormone treatments

  • Analyze complex composition changes using Blue Native PAGE followed by immunoblotting

  • Correlate with physiological responses

These approaches help elucidate how ethylene signaling through EIN4 integrates with other hormone pathways to coordinate plant responses .

What are the most effective approaches for using EIN4 antibodies in plant tissue imaging studies?

For optimal imaging of EIN4 in plant tissues:

• Sample preparation optimization:

  • Fixation: Test aldehydes (4% paraformaldehyde, glutaraldehyde) and compare with freeze substitution methods

  • Embedding: Paraffin for general histology, LR White resin for better epitope preservation

  • Sectioning: 5-10 μm sections for light microscopy, 70-100 nm for electron microscopy

  • Antigen retrieval: Compare heat-induced, enzymatic, and pH-based methods

• Immunolabeling strategies:

  • Direct methods: Directly labeled primary antibodies for simple, one-step detection

  • Indirect methods: Primary EIN4 antibody followed by labeled secondary antibody for signal amplification

  • Signal enhancement: Tyramide signal amplification for low-abundance detection

  • Multiplex labeling: Combine with antibodies against other proteins of interest using different fluorophores

• Advanced imaging techniques:

  • Confocal microscopy: For co-localization studies with other proteins

  • Super-resolution microscopy: STORM or PALM for nanoscale localization

  • Immunogold electron microscopy: For ultrastructural localization

  • Live-cell imaging: Using minimally invasive labeling techniques

• Quantitative analysis:

  • Measure signal intensity across tissues/cells

  • Analyze co-localization coefficients

  • Track changes in distribution following treatments

The choice of method depends on the specific research question, with consideration for the subcellular localization of EIN4 in the endoplasmic reticulum membrane .

How can bispecific antibody technologies be applied to study EIN4 interaction with other ethylene receptors?

Bispecific antibody technologies offer powerful approaches for investigating EIN4's interactions:

• Generation of bispecific antibodies:

  • Knobs-into-holes technology: Successfully applied to both IgG1 and IgG4 isotypes

  • Design considerations: Target epitopes on non-interactive domains of receptors

  • Production methods: Similar efficiency and quality can be achieved as with standard antibodies

  • Validation: Confirm binding to both targets individually and simultaneously

• Applications in receptor interaction studies:

  • Co-localization analysis: Use bispecific antibodies targeting EIN4 and another receptor (e.g., ETR1)

  • Conformational change detection: Design bispecific antibodies that only bind when receptors are in specific conformational states

  • Functional modulation: Create bispecific antibodies that force or prevent heterodimerization

• Quantitative measurements:

  • Flow cytometry to quantify receptor interactions in protoplasts

  • FRET-based assays using labeled bispecific antibodies

  • Surface plasmon resonance for interaction kinetics

• In vivo applications:

  • Microinjection of bispecific antibodies into plant cells

  • Use cell-penetrating peptides for antibody delivery

  • Create plant-expressible recombinant antibody fragments

This emerging technology could provide unprecedented insights into the dynamics and functional significance of ethylene receptor interactions .

How can I resolve weak or absent signal problems when using EIN4 antibodies in Western blots?

When facing weak or absent signals in EIN4 Western blots:

Additionally, membrane proteins like EIN4 may require:

• Extended denaturation time in sample buffer

• Lower temperature transfer conditions

• Addition of 0.1% SDS to transfer buffer

• Use of specialized extraction buffers for membrane proteins

What strategies can address cross-reactivity issues when studying closely related ethylene receptors?

When studying EIN4 among the five Arabidopsis ethylene receptors, cross-reactivity can be addressed through:

• Antibody selection and optimization:

  • Choose antibodies raised against unique regions of EIN4

  • Test antibodies against recombinant proteins of all five receptors

  • Perform peptide competition assays with EIN4-specific peptides

• Genetic approaches:

  • Use receptor knockout lines as negative controls

  • Employ transgenic lines with epitope-tagged receptors

  • Create lines with single receptors complementing quintuple mutants

• Technical refinements:

  • Increase washing stringency (higher salt, detergent concentration)

  • Pre-absorb antibodies with recombinant proteins of other receptors

  • Use monoclonal rather than polyclonal antibodies when possible

• Data validation:

  • Compare antibody-based results with genetic evidence

  • Confirm key findings with orthogonal techniques

  • Use multiple antibodies targeting different EIN4 epitopes

• Advanced specificity testing:

  • Employ biophysics-informed modeling similar to that used for customized antibody specificity profiles

  • Consider methods used in other receptor families for disentangling binding modes of closely related epitopes

These approaches require significant investment but yield more reliable results when working with closely related proteins .

How can the functionality of EIN4 antibodies be preserved during long-term storage and repeated use?

To maintain EIN4 antibody functionality:

• Storage recommendations:

  • Aliquot antibodies in small volumes (10-50 μl) upon receipt

  • Store at -20°C for short-term or -80°C for long-term storage

  • Add stabilizing proteins (BSA, 0.1%) if not already present

  • Include preservatives (sodium azide, 0.02%) for refrigerated storage

• Handling practices:

  • Avoid repeated freeze-thaw cycles (more than 5)

  • Thaw on ice and return to storage promptly

  • Centrifuge briefly after thawing to collect solution

  • Use sterile technique to prevent contamination

• Monitoring antibody quality:

  • Maintain a reference sample for comparative testing

  • Document signal intensity across usage time

  • Test with positive controls before critical experiments

  • Monitor background levels as indicator of degradation

• Regeneration of immunoblotting membranes:

  • Stripping should be gentle (commercial stripping buffers or 0.2M glycine, pH 2.5)

  • Limit number of stripping cycles (2-3 maximum)

  • Re-block thoroughly after stripping

  • Store stripped membranes at 4°C in TBS with 0.02% sodium azide

• Alternative to antibody regeneration:

  • Cut membranes to probe different regions with different antibodies

  • Use multiplex fluorescent detection systems

  • Sequential probing without stripping for non-overlapping proteins

These practices maximize antibody lifespan and ensure consistent performance in research applications .

How might single-cell proteomics approaches enhance EIN4 antibody-based research?

Single-cell proteomics offers exciting potential for EIN4 research:

• Technical approaches for plant single-cell proteomics:

  • Protoplast isolation followed by microfluidic separation

  • Laser capture microdissection of specific cell types

  • Fluorescence-activated cell sorting based on cell-type specific markers

  • Nanobody-based proximity labeling for subcellular resolution

• EIN4 receptor quantification at single-cell level:

  • Mass cytometry (CyTOF) with metal-labeled EIN4 antibodies

  • Single-cell Western blotting technologies

  • Antibody-based microarray platforms

  • Digital ELISA platforms with single-molecule sensitivity

• Data analysis considerations:

  • Computational approaches for sparse data integration

  • Trajectory analysis to identify developmental regulation

  • Correlation with single-cell transcriptomics

  • Spatial reconstruction of tissue-level patterns

• Potential research insights:

  • Cell-type specific EIN4 expression patterns

  • Heterogeneity in receptor levels between apparently identical cells

  • Correlation between receptor abundance and downstream signaling

  • Dynamic changes during development and stress responses

These approaches would provide unprecedented resolution of ethylene receptor dynamics across different cell types .

What are the prospects for developing conformation-specific EIN4 antibodies to study receptor activation states?

Developing conformation-specific antibodies for EIN4 represents an advanced frontier:

• Strategic approaches:

  • Target regions known to undergo conformational changes (based on structural data from ETR1)

  • Focus on the D25 equivalent region in EIN4, which is critical for ethylene binding in ETR1

  • Design specific immunization strategies using:

    • Peptides constrained in active/inactive conformations

    • Receptors locked in specific states via mutations

    • Receptor fragments crystallized in defined conformations

• Validation methodologies:

  • Test antibody binding to wild-type vs. constitutively active mutants

  • Compare binding in presence/absence of ethylene

  • Use hydrogen-deuterium exchange mass spectrometry to confirm conformational epitopes

  • Employ molecular dynamics simulations to guide epitope selection

• Potential applications:

  • Real-time monitoring of receptor activation in living tissues

  • Quantification of active vs. inactive receptor populations

  • Screening for compounds that modulate receptor conformations

  • Investigating receptor cross-regulation mechanisms

• Technical challenges:

  • Preserving native membrane protein conformations during immunization

  • Maintaining conformational epitopes during sample preparation

  • Distinguishing specific conformational changes from non-specific effects

  • Standardizing assay conditions for reproducible results

Similar approaches have revolutionized studies of G-protein coupled receptors and could transform our understanding of ethylene perception mechanisms .

How might machine learning approaches enhance the design and application of EIN4-specific antibodies?

Machine learning offers promising avenues for EIN4 antibody research:

• Epitope prediction and antibody design:

  • Deep learning models can predict optimal epitopes for generating receptor-specific antibodies

  • Sequence-based algorithms can identify regions of maximum divergence between ethylene receptors

  • Structure-based approaches can target surface-exposed, receptor-specific domains

  • Biophysics-informed models similar to those used for customized antibody specificity profiles

• Optimizing experimental conditions:

  • Machine learning can identify patterns in successful vs. unsuccessful experiments

  • Bayesian optimization for efficiently determining optimal antibody concentrations

  • Neural networks to predict cross-reactivity based on sequence homology

  • Random forest algorithms to identify critical variables in immunoprecipitation success

• Image analysis enhancements:

  • Convolutional neural networks for automated detection of staining patterns

  • Segmentation algorithms for quantifying receptor distribution

  • Classification of cellular phenotypes based on receptor localization

  • Super-resolution image reconstruction

• Integration with multi-omics data:

  • Correlate antibody-based protein detection with transcriptomics

  • Predict protein-protein interactions based on co-localization patterns

  • Model receptor dynamics in different genetic backgrounds

  • Systems biology approaches to place EIN4 in broader signaling networks

These computational approaches could significantly accelerate discovery and improve reproducibility in EIN4 antibody research .