ERL2 Antibody

What is ERL2 and why is it significant in plant research?

ERL2 is a receptor-like kinase belonging to the ERECTA family in plants. It contains extracellular leucine-rich repeat (LRR) domains, a transmembrane region, and an intracellular kinase domain. This receptor plays important roles in plant development and immunity, functioning redundantly with other family members like ERECTA (ER) and ERL1.

In Arabidopsis, ERL2 contributes to the regulation of:

• Stomatal development and patterning

• Longitudinal growth of aboveground organs

• Shoot apical meristem regulation

• Immune responses against pathogens such as necrotrophic fungi

Research has demonstrated that while single erl2 mutants may not display obvious phenotypes, double and triple mutant combinations with er and erl1 show enhanced susceptibility to pathogens like PcBMM, indicating functional redundancy within this receptor family .

How does ERL2 differ structurally from other ERECTA family members?

• The extracellular LRR domains contain sequence variations that may affect ligand specificity

• The precise arrangement of LRR repeats differs subtly between family members

• Certain key residues in the kinase domain show variability that may impact signaling specificity

These structural differences have functional implications, as indicated by research showing that specific extracellular LRR domains (such as the 10th and 18th LRRs) have differential importance in immune responses versus developmental functions .

What technical challenges are associated with ERL2 antibody production?

Producing specific antibodies against ERL2 presents several technical challenges:

• High sequence homology with other ERECTA family members (ER and ERL1) increases the risk of cross-reactivity

• The membrane-bound nature of ERL2 makes it difficult to express and purify for immunization

• Post-translational modifications may affect epitope recognition

• Maintaining the native conformation of ERL2 during antibody production is challenging

• Low natural expression levels in many tissues may complicate validation

These challenges parallel issues encountered with other receptor antibodies, such as those targeting estrogen receptor beta, where inadequate validation has led to conflicting results in the field .

What validation methods are essential for confirming ERL2 antibody specificity?

Rigorous validation of ERL2 antibodies requires a comprehensive approach:

• Genetic controls: Test antibodies on wild-type plants versus erl2 knockout mutants

• Overexpression systems: Evaluate antibody performance in plants overexpressing ERL2

• Western blotting: Confirm detection of a single band of appropriate molecular weight

• Immunoprecipitation followed by mass spectrometry (IP-MS): Verify the identity of captured proteins

• Cross-reactivity testing: Assess binding to related proteins (ER, ERL1) in knockout/overexpression lines

• Multiple application testing: Validate performance across different techniques (WB, IHC, IP)

Recent studies on receptor antibodies highlight that many commercially available antibodies fail these validation steps, emphasizing the importance of thorough validation before experimental use .

How should researchers optimize immunohistochemistry protocols for ERL2 detection in plant tissues?

Optimizing immunohistochemistry for ERL2 detection requires attention to several key factors:

• Fixation method: Test different fixatives (e.g., paraformaldehyde, glutaraldehyde) to preserve epitope accessibility

• Antigen retrieval: Evaluate various antigen retrieval methods to unmask epitopes

• Blocking optimization: Determine optimal blocking conditions to reduce background (typically 3-5% BSA or normal serum)

• Antibody dilution: Test a range of dilutions to find the optimal signal-to-noise ratio

• Incubation conditions: Optimize temperature and duration for primary antibody incubation

• Detection system: Compare fluorescent versus chromogenic detection methods

• Controls: Include both wild-type and erl2 mutant tissues in each experiment

Similar approaches have been necessary for successful detection of other plant receptor proteins in tissue samples .

What are the recommended approaches for co-immunoprecipitation studies involving ERL2?

For effective co-immunoprecipitation of ERL2 and interacting partners:

• Membrane protein extraction: Use mild detergents (0.5-1% NP-40, digitonin, or DDM) that preserve protein-protein interactions

• Buffer optimization: Include stabilizing agents (glycerol, specific ions) in extraction and wash buffers

• Crosslinking consideration: For transient interactions, mild crosslinking may be necessary

• Pre-clearing: Implement stringent pre-clearing steps to reduce non-specific binding

• Antibody immobilization: Compare direct coupling versus protein A/G approaches

• Elution strategies: Test various elution methods to maximize recovery while maintaining complex integrity

• Interacting protein detection: Use specific antibodies against suspected partners or MS analysis

These methodological considerations are crucial when investigating how ERL2 forms complexes with other RLKs and RLPs involved in developmental and immune signaling pathways .

How can researchers address contradictory results between different ERL2 antibodies?

When different ERL2 antibodies yield conflicting results, consider this systematic approach:

• Epitope mapping: Identify the specific epitopes recognized by each antibody

• Validation rigor: Re-evaluate each antibody using multiple validation methods

• Functional domain analysis: Assess whether antibodies target functional domains that may be masked in protein complexes

• Post-translational modifications: Determine if modifications affect epitope accessibility

• Complementary methods: Employ non-antibody techniques to resolve contradictions

• Quantitative comparison: Use quantitative metrics to compare specificity and sensitivity

This systematic approach mirrors the strategy used to resolve contradictions in other receptor antibody research, where comprehensive validation revealed that only one of thirteen tested antibodies was truly specific .

What metrics should be used to quantitatively assess ERL2 antibody performance?

Quantitative assessment of ERL2 antibody performance should include:

These quantitative metrics provide objective means to compare different antibodies and ensure experimental reliability .

How should researchers interpret ERL2 localization patterns in relation to known functions?

When interpreting ERL2 localization data:

• Correlate with developmental context: ERL2 expression patterns change during development, affecting interpretation

• Consider redundancy: ERL2 often functions redundantly with ER and ERL1, so co-localization studies are important

• Relate to known mutant phenotypes: Connect localization to functional data from genetic studies

• Compare with transcript data: Verify protein localization against transcript expression patterns

• Assess in response to stimuli: Examine how localization changes during immune responses or developmental transitions

Research has shown that ERECTA family proteins, including ERL2, function in various tissues and developmental contexts, often with overlapping but distinct patterns that relate to their redundant yet specialized functions .

How can ERL2 antibodies be used to study receptor complex formation?

To investigate ERL2 receptor complex formation:

• Sequential immunoprecipitation: Use anti-ERL2 antibodies followed by antibodies against potential partners

• Blue native PAGE: Combine with immunoblotting to identify native complex sizes

• Density gradient analysis: Separate complexes based on size/density followed by immunodetection

• In situ proximity ligation: Visualize protein interactions in intact tissues

• FRET/FLIM analysis: Complement antibody studies with fluorescence-based interaction assays

These approaches can help elucidate how ERL2 forms different complexes with proteins like TMM, ER, ERL1, and other signaling components in different developmental or immune contexts .

What role do ERL2 antibodies play in understanding the differential functions of ERL2 LRR domains?

ERL2 antibodies can provide insights into LRR domain functions through:

• Domain-specific antibodies: Developing antibodies targeting specific LRR regions

• Epitope masking experiments: Using antibodies to block specific LRR domains and assess functional consequences

• Conformational antibodies: Detecting activation-dependent conformational changes

• Interaction mapping: Identifying which LRR domains mediate specific protein-protein interactions

• Structure-function correlation: Relating antibody binding to functional outcomes

Research has already demonstrated that specific LRR domains, such as the 10th and 18th LRRs, have differential importance in immune versus developmental functions, with the 18th LRR being crucial for immunity while the 10th LRR appears more important for developmental processes .

How can phospho-specific ERL2 antibodies contribute to signaling pathway analysis?

Phospho-specific ERL2 antibodies enable detailed analysis of signaling dynamics:

• Activation kinetics: Monitor the timing of ERL2 phosphorylation following stimulus

• Pathway crosstalk: Examine how different stimuli affect phosphorylation patterns

• Subcellular analysis: Track where in the cell ERL2 phosphorylation occurs

• Quantitative signaling: Measure the proportion of phosphorylated versus total ERL2

• Phosphorylation site mapping: Identify which residues are phosphorylated under different conditions

This approach would complement genetic studies showing that ERL2 functions redundantly with ER and ERL1 in immune responses, potentially revealing differences in activation patterns that explain their overlapping yet distinct functions .

What are common causes of non-specific binding with ERL2 antibodies and how can they be addressed?

Common causes of non-specific binding and their solutions include:

Similar issues have been observed with other receptor antibodies, where extensive optimization was required to achieve specificity .

How should storage conditions be optimized to maintain ERL2 antibody functionality?

To preserve ERL2 antibody functionality:

• Storage temperature: Store at -20°C or -80°C as recommended by manufacturer

• Aliquoting strategy: Prepare single-use aliquots to avoid freeze-thaw cycles

• Buffer composition: Verify optimal buffer components (glycerol, protein stabilizers)

• Contamination prevention: Use sterile technique when handling

• Periodic validation: Re-validate antibody performance after extended storage

• Documentation: Keep detailed records of performance over time

Research has shown that antibody functionality can deteriorate over time, as demonstrated with the 14C8 antibody that lost its ability to distinguish between positive and negative controls after extended storage .

What strategies can overcome poor signal detection when using ERL2 antibodies?

To improve weak signal detection with ERL2 antibodies:

• Sample preparation optimization:

  • Use optimized extraction buffers for membrane proteins

  • Concentrate samples if protein abundance is low

  • Consider enrichment methods for membrane fractions

• Signal amplification techniques:

  • Employ tyramide signal amplification for immunohistochemistry

  • Use high-sensitivity detection reagents for Western blotting

  • Consider biotin-streptavidin systems for signal enhancement

• Instrumentation adjustments:

  • Increase exposure times while monitoring background

  • Use more sensitive detection systems (e.g., chemiluminescence vs. colorimetric)

  • Optimize imaging parameters for weak signals

• Antibody modifications:

  • Try different concentrations of primary antibody

  • Extend incubation times at lower temperatures

  • Consider different detection antibodies with higher sensitivity

These approaches can help detect low-abundance ERL2 protein while maintaining specificity .

How might single-molecule techniques advance ERL2 antibody applications?

Single-molecule techniques offer promising advances for ERL2 research:

• Single-molecule localization microscopy (PALM/STORM) applications:

  • Super-resolution imaging of ERL2 distribution in membranes

  • Quantitative analysis of receptor clustering

  • Co-localization studies at nanometer precision

• Single-molecule tracking:

  • Monitoring ERL2 dynamics in living cells

  • Measuring diffusion rates in different membrane domains

  • Tracking conformational changes upon ligand binding

• Single-molecule pull-down (SiMPull):

  • Direct visualization of protein-protein interactions

  • Determination of complex stoichiometry

  • Analysis of heterogeneity in protein complexes

These technologies would provide unprecedented insights into how ERL2 functions in concert with other ERf members in various signaling contexts .

What new validation approaches might ensure greater ERL2 antibody specificity?

Emerging validation approaches for enhancing ERL2 antibody specificity include:

• CRISPR-engineered validation systems:

  • Endogenous tagging of ERL2 for antibody validation

  • Creation of domain-specific deletions to map epitopes

  • Generation of comprehensive knockout lines as negative controls

• Microfluidic antibody characterization:

  • High-throughput epitope mapping

  • Rapid assessment of cross-reactivity profiles

  • Automated optimization of binding conditions

• Structural biology integration:

  • Using structural data to predict and avoid cross-reactive epitopes

  • Structure-guided antibody engineering

  • Computational prediction of optimal antigenic regions

• Recombinant antibody technologies:

  • Development of highly specific recombinant antibodies

  • Affinity maturation for improved specificity

  • Creation of antibody panels targeting different ERL2 epitopes

These approaches could significantly improve antibody validation standards, addressing issues similar to those identified in other receptor antibody fields .

How might proteogenomic approaches enhance ERL2 antibody development and validation?

Proteogenomic strategies for improved ERL2 research include:

• Integrated transcript-protein expression analysis:

  • Correlation of ERL2 transcript and protein levels across tissues

  • Identification of post-transcriptional regulation

  • Prediction of protein abundance for antibody sensitivity requirements

• Epitope conservation analysis:

  • Cross-species comparison of ERL2 sequences

  • Identification of conserved vs. variable regions

  • Selection of epitopes with optimal specificity/conservation balance

• Machine learning applications:

  • Prediction of optimal antigenic regions

  • Forecasting cross-reactivity risks

  • Automated validation data analysis

• Multi-omics validation pipelines:

  • Integration of transcriptomics, proteomics, and antibody-based detection

  • Systems-level confirmation of antibody specificity

  • Comprehensive documentation of validation evidence

These approaches would provide more robust foundations for antibody development, potentially avoiding the pitfalls identified in other receptor antibody fields where inadequate validation led to conflicting results .