ERECTA Antibody

What is ERECTA and why is it important in plant molecular research?

ERECTA (ER) is a receptor-like kinase in Arabidopsis thaliana that plays crucial roles in various developmental processes, including seed size determination and integument cell proliferation. ERECTA contains an extracellular domain, a transmembrane domain, and intracellular cytoplasmic domains that are essential for its function. Several ERECTA mutants have been characterized, including er563, er795, and er1214, which contain premature stop codons resulting in truncated proteins of varying lengths . ERECTA signaling is regulated by U-box ubiquitin E3 ligases, specifically PUB30 and PUB31, which directly interact with ERECTA and attenuate its signaling pathway . Due to its importance in plant development, ERECTA has become a significant focus in plant molecular biology research.

What experimental validation must be performed before using an ERECTA antibody?

Before using an ERECTA antibody in experiments, researchers should perform comprehensive validation to ensure reliability. Similar to general antibody validation principles, ERECTA antibody characterization should document: (i) that the antibody binds specifically to ERECTA protein; (ii) that the antibody recognizes ERECTA when present in complex protein mixtures such as plant cell lysates; (iii) that the antibody does not cross-react with proteins other than ERECTA; and (iv) that the antibody performs consistently under the specific experimental conditions of planned assays .

For ERECTA antibody validation, researchers should:

• Confirm binding to recombinant ERECTA protein using ELISA

• Verify specificity using Western blot analysis with wild-type and erecta mutant tissues

• Test antibody performance in planned applications (immunoprecipitation, immunohistochemistry)

• Include appropriate negative controls (erecta null mutants) and positive controls

What are the common experimental applications for ERECTA antibodies?

ERECTA antibodies are employed in various experimental approaches to study ERECTA function and regulation:

The choice of application depends on the specific research question being addressed and the characteristics of the available ERECTA antibody.

How should ERECTA antibody experiments be controlled and optimized?

Proper controls are essential for ERECTA antibody experiments. For plant studies, include:

• Genetic controls: Compare wild-type plants with erecta null mutants (e.g., er-105) . The absence of signal in mutants confirms antibody specificity.

• Expression controls: Use plants expressing tagged ERECTA proteins (e.g., ERECTA-FLAG) to validate antibody performance against known expression patterns .

• Loading controls: Include detection of a constitutively expressed protein (e.g., actin) to normalize protein loading in quantitative analyses .

• Treatment controls: When studying ERECTA regulation, include appropriate controls such as untreated samples alongside proteasome inhibitor treatments (e.g., MG132) .

For optimization, researchers should test multiple antibody concentrations, incubation times, and buffer conditions to determine optimal signal-to-noise ratios for each experimental approach.

How can ERECTA antibodies be used to investigate receptor ubiquitination and degradation?

ERECTA undergoes ubiquitination and subsequent degradation via the 26S proteasome pathway as a key regulatory mechanism. To study this process:

• Ubiquitination analysis: Perform immunoprecipitation of ERECTA using anti-ERECTA or anti-tag antibodies (if using tagged ERECTA), followed by immunoblotting with anti-ubiquitin antibodies. This approach allows detection of ubiquitinated forms of ERECTA .

• Quantification: Calculate the relative signal intensity ratio between ubiquitinated ERECTA (detected by anti-ubiquitin antibodies) and immunoprecipitated ERECTA (detected by anti-ERECTA or anti-tag antibodies) to assess ubiquitination levels .

• Proteasome inhibition: Treat samples with proteasome inhibitors (e.g., 20μM MG132 for 48 hours) to prevent degradation of ubiquitinated ERECTA, which facilitates detection of these modified forms. Compare protein levels before and after inhibitor treatment to assess degradation rates .

• Protein stability assays: Compare ERECTA protein accumulation in different genetic backgrounds (e.g., wild-type versus pub30 pub31 mutants) while normalizing to transcript levels to distinguish post-translational effects from transcriptional regulation .

For accurate interpretation, it's crucial to verify that changes in ubiquitination are not due to alterations in ERECTA transcript levels, which can be assessed using techniques such as qRT-PCR .

What are the most effective methods for studying ERECTA's protein-protein interactions?

Multiple complementary approaches should be used to comprehensively characterize ERECTA's protein-protein interactions:

• Yeast Two-Hybrid (Y2H): Use a truncated ERECTA protein with the cytosolic domain (ERECTA_CD) as bait to screen for potential interacting partners. This method identified direct interactions between ERECTA_CD and PUB30/31 .

• In vitro pull-down assays: Confirm Y2H results using purified recombinant proteins. For ERECTA studies, this involves expressing and purifying ERECTA_CD and potential interacting proteins with affinity tags (GST, MBP), then performing pull-down experiments to verify direct physical interactions .

• Biolayer Interferometry (BLI): Quantitatively characterize the kinetics of protein-protein interactions and determine binding affinities. BLI analysis of ERECTA with PUB30/31 revealed micromolar affinity interactions, suggesting transient and dynamic associations .

• Co-Immunoprecipitation (Co-IP): Verify interactions in vivo by immunoprecipitating ERECTA from plant tissues and detecting associated proteins by immunoblotting. This technique is particularly useful for studying ligand-induced interactions, as demonstrated with EPFL6 peptide treatment enhancing ERECTA association with other proteins .

When studying receptor complexes, consider ligand-induced changes in interaction dynamics by comparing protein associations before and after treatment with signaling peptides or other activators.

How can ERECTA antibodies help investigate co-receptor dynamics and complex formation?

ERECTA functions in complex with co-receptors like BAK1, with complex formation often triggered by ligands. To study these dynamics:

• Sequential Co-IP: Immunoprecipitate ERECTA first, then probe for co-receptors like BAK1 in the precipitated material. Compare samples with and without ligand treatment (e.g., EPFL6 peptide) to assess ligand-induced complex formation .

• Reciprocal Co-IP: Immunoprecipitate the co-receptor (e.g., BAK1) and probe for ERECTA to confirm the interaction from both perspectives.

• Proximity labeling: Use antibodies to validate results from proximity labeling approaches that identify proteins in close physical proximity to ERECTA in vivo.

• Mutant analysis: Compare receptor complex formation in various genetic backgrounds (e.g., wild-type versus pub30 pub31 mutants) to understand factors affecting complex stability .

When analyzing co-receptor dynamics, it's important to note that some interactions may be weak or transient in the absence of ligand stimulation, as observed with BAK1 being weakly detected without peptide treatment but strongly associated with PUB30/31 after EPFL6 peptide incubation .

What considerations are important when using ERECTA antibodies for analysis of truncated or mutant ERECTA proteins?

When studying ERECTA mutants with antibodies, several important considerations apply:

• Epitope availability: Determine whether the antibody's epitope is present in the mutant protein. For instance, in erecta mutants like er563 and er795, which produce severely truncated proteins (124 and 99 amino acids, respectively), antibodies targeting the C-terminal region would not detect these proteins .

• Domain-specific antibodies: Consider using domain-specific antibodies. For mutants like er1214, which retains the extracellular and transmembrane domains but lacks cytoplasmic domains, antibodies targeting different regions would yield different results .

• Tagged constructs: For complex analyses, use epitope-tagged ERECTA constructs (e.g., ERECTA-FLAG) in complementation experiments with erecta null mutants. This approach allows consistent detection regardless of mutations in the native protein .

• Validation in mutant backgrounds: Always validate antibody performance in various mutant backgrounds to ensure reliable interpretation of results.

A comprehensive experimental design would include parallel analysis with both N-terminal and C-terminal targeting antibodies to distinguish between truncation effects and complete absence of the protein.

What are common technical issues with ERECTA antibodies and how can they be resolved?

Researchers may encounter several technical challenges when working with ERECTA antibodies:

• Low signal strength: This may occur due to low ERECTA abundance or expression. Solutions include:

  • Increasing protein concentration in samples

  • Optimizing extraction buffers to improve protein solubilization

  • Using tagged ERECTA constructs under native promoters to facilitate detection

  • Enriching samples through immunoprecipitation before detection

• High background: Non-specific binding can complicate interpretation. Address by:

  • Increasing blocking concentration and time

  • Adding detergents in washing steps

  • Pre-adsorbing antibodies against extracts from erecta null mutants

  • Validating specificity through parallel analysis of erecta null mutant samples

• Inconsistent results: Variability between experiments may stem from:

  • Antibody batch variations (use consistent lots when possible)

  • Variations in protein extraction efficiency

  • Developmental or environmental factors affecting ERECTA expression

  • Post-translational modifications changing epitope accessibility

For detecting low-abundance proteins like ERECTA, consider signal amplification methods or highly sensitive detection systems like chemiluminescence with extended exposure times .

How should sample preparation be optimized for ERECTA antibody-based experiments?

Effective sample preparation is crucial for successful ERECTA antibody experiments:

• Growth conditions: Standardize plant growth conditions (temperature, light, media composition) to minimize variations in ERECTA expression. For Arabidopsis seedlings, growth at 22°C on half-strength Murashige and Skoog (MS) medium for 5 days provides consistent material for analysis .

• Tissue selection: Choose appropriate tissues based on ERECTA expression patterns. Consider developmental stages carefully, as ERECTA and its regulatory proteins like PUB30/31 may show overlapping expression in specific tissues such as developing cotyledon epidermis .

• Protein extraction: Use buffers containing protease inhibitors to prevent degradation. For studies of post-translationally modified ERECTA, include specific inhibitors:

  • Proteasome inhibitors (e.g., MG132) to prevent degradation of ubiquitinated forms

  • Phosphatase inhibitors when studying ERECTA phosphorylation

  • Deubiquitinating enzyme inhibitors when analyzing ubiquitination patterns

• Sample handling: Process samples quickly and maintain cold temperatures throughout extraction to minimize protein degradation. For treatments like proteasome inhibition, consistent application methods are essential (e.g., transferring seedlings to liquid half-strength MS medium with 20μM MG132 for 48 hours) .

Standardizing these procedures across experiments will greatly improve reproducibility and reliability of results.

What advanced techniques are emerging for improved ERECTA antibody specificity and sensitivity?

Several advanced approaches can enhance ERECTA antibody experiments:

• Recombinant antibody technology: The development of recombinant antibodies against ERECTA could provide more consistent reagents with defined specificity, similar to efforts in human protein antibody development .

• Epitope mapping: Detailed characterization of antibody epitopes helps predict cross-reactivity and binding to mutant forms of ERECTA. This information is particularly valuable when studying truncated ERECTA variants .

• Multiparameter analysis: Combining antibody-based detection with other analytical techniques:

  • Correlating protein detection (via antibodies) with transcript analysis (via RT-PCR)

  • Using antibodies in conjunction with mass spectrometry for identification of post-translational modifications

  • Combining immunoprecipitation with sequencing technologies to identify DNA/RNA binding

• Microarray-based validation: High-throughput characterization of antibody specificity against protein arrays containing potential cross-reactive proteins from the same family as ERECTA could improve confidence in experimental results.

These approaches align with broader efforts in the scientific community to enhance antibody characterization for improved research reproducibility .

How can researchers effectively document and share ERECTA antibody characterization data?

Proper documentation and sharing of ERECTA antibody characterization is essential for research reproducibility:

• Comprehensive reporting: Document all validation experiments performed, including:

  • Antibody source, catalog number, and lot number

  • Complete characterization data demonstrating specificity

  • Detailed protocols for each application (Western blot, IP, etc.)

  • Images of complete blots including molecular weight markers

• Standardized metadata: Use standardized formats for antibody information following guidelines similar to those from antibody validation initiatives like Antibodypedia or the Developmental Studies Hybridoma Bank (DSHB) .

• Use of identifiers: Implement Research Resource Identifiers (RRIDs) for ERECTA antibodies to enable consistent tracking in the literature .

• Repository submission: Consider submitting detailed characterization data to antibody validation repositories to benefit the broader research community, similar to approaches taken by the NeuroMab initiative for neuronal antibodies .

• Method transparency: In publications, provide sufficiently detailed methods for antibody-based experiments to allow reproduction, including:

  • Complete immunoblot protocols with buffer compositions

  • Antibody dilutions and incubation conditions

  • Image acquisition parameters and analysis methods

This comprehensive documentation approach aligns with broader efforts to enhance reproducibility in antibody-based research across scientific disciplines .

How should researchers interpret contradictory results from different ERECTA antibody-based assays?

When faced with contradictory results from different ERECTA antibody-based assays, researchers should consider:

• Assay-specific factors: Different applications (Western blot, immunoprecipitation, immunohistochemistry) have distinct requirements for antibody performance. An antibody that works well in one application may fail in another due to differences in protein conformation, epitope accessibility, or experimental conditions .

• Epitope accessibility: Post-translational modifications, protein-protein interactions, or conformational changes may mask epitopes in some experimental contexts but not others. Consider using multiple antibodies targeting different ERECTA regions to provide complementary information.

• Cross-reactivity profiles: Antibodies may exhibit different cross-reactivity patterns depending on the experimental conditions. Validate specificity in each experimental system using appropriate controls, particularly erecta null mutants .

• Systematic validation approach:

  • Compare results with orthogonal methods not relying on antibodies

  • Perform genetic validation using mutants and complementation lines

  • Conduct domain-specific analyses using truncated ERECTA variants

  • Test multiple independently derived antibodies against the same target

• Control experiments: When comparing ERECTA detection across different conditions, include critical controls:

  • Transcript analysis to distinguish protein-level from transcriptional effects

  • Parallel analysis of wild-type and erecta mutant samples in each experimental run

  • Appropriate treatments (e.g., proteasome inhibitors) to control for protein stability effects

What experimental designs can differentiate between direct and indirect effects in ERECTA antibody studies?

To distinguish direct from indirect effects in ERECTA signaling and regulation:

• In vitro reconstitution: Use purified components to demonstrate direct interactions. Techniques like in vitro pull-down assays with recombinant ERECTA_CD and potential interacting proteins can confirm direct physical interactions without cellular context .

• Quantitative binding assays: Employ biolayer interferometry (BLI) to characterize direct protein-protein interaction kinetics and determine binding affinities, as demonstrated for ERECTA interactions with PUB30/31 .

• Domain mapping: Identify specific domains required for interactions through deletion and point mutation analysis. This approach helped characterize the interaction between ERECTA's cytosolic domain and regulatory proteins .

• Sequential dependency analysis: Use genetic approaches with various mutant combinations to establish the sequence of events in signaling pathways. For example, comparing ERECTA protein levels in wild-type versus pub30 pub31 backgrounds while monitoring transcript levels helped establish post-translational regulation .

• Inducible systems: Implement temporally controlled expression or activity systems to differentiate between immediate (likely direct) and delayed (possibly indirect) effects following ERECTA activation or inhibition.

• Cross-linking studies: Employ protein cross-linking followed by mass spectrometry to identify direct binding partners of ERECTA in vivo, which can be validated using antibody-based methods.

This multi-faceted approach provides robust evidence for distinguishing direct interactions from indirect regulatory relationships in ERECTA signaling networks.

How can researchers effectively design experiments to study ERECTA post-translational modifications?

Post-translational modifications (PTMs) of ERECTA, particularly ubiquitination, are critical regulatory mechanisms. To study these effectively:

• PTM-specific detection strategies:

  • For ubiquitination: Immunoprecipitate ERECTA, then probe with anti-ubiquitin antibodies

  • For phosphorylation: Use phospho-specific antibodies or phospho-enrichment followed by mass spectrometry

  • For other modifications: Consider specialized enrichment methods before antibody detection

• Quantitative analysis:

  • Calculate the relative ratio of modified to unmodified ERECTA

  • Compare modification levels across different genetic backgrounds and treatments

  • Normalize appropriately to total ERECTA levels to account for expression differences

• Modification site mapping:

  • Generate site-specific mutants by replacing modifiable residues

  • Compare wild-type and mutant ERECTA behavior in vivo

  • Use mass spectrometry to identify specific modification sites

• Temporal dynamics:

  • Implement time-course experiments following ligand stimulation

  • Use modification-specific antibodies to track changes over time

  • Consider pulse-chase approaches to study modification turnover rates

• Pharmacological interventions:

  • Use specific inhibitors of modification processes (e.g., MG132 for proteasome inhibition)

  • Compare modification patterns before and after inhibitor treatment

  • Combine inhibitor treatments with genetic approaches for comprehensive analysis

This experimental framework provides a robust approach to characterize the complex post-translational regulation of ERECTA in plant development and signaling.

How can ERECTA antibodies be leveraged for studying receptor trafficking and membrane dynamics?

ERECTA receptor trafficking and membrane dynamics represent important aspects of its regulation. Researchers can employ several approaches:

• Subcellular fractionation: Use ERECTA antibodies to detect the receptor in different cellular compartments following biochemical fractionation. Compare receptor distribution across conditions to track trafficking patterns.

• Endocytosis assays: Combine surface biotinylation or external epitope tagging with ERECTA antibodies to monitor internalization rates. This approach can reveal how regulatory proteins like PUB30/31 might influence receptor endocytosis following ligand binding.

• Co-localization studies: Use ERECTA antibodies in combination with markers for different endocytic compartments to track the receptor's itinerary through the endocytic pathway. This is particularly relevant when studying how ubiquitination by PUB30/31 may affect ERECTA trafficking .

• Protease protection assays: Determine the topology and membrane insertion of ERECTA using protease treatment of intact or permeabilized cells followed by detection with domain-specific antibodies.

• Receptor recycling vs. degradation: Use antibodies to distinguish between recycling and degradative pathways by quantifying ERECTA in different compartments following ligand stimulation. Comparing wild-type and pub30 pub31 mutants could reveal how ubiquitination influences trafficking decisions .

These approaches can provide crucial insights into how ERECTA localization and movement contribute to its signaling functions in plant development.

What emerging technologies might enhance ERECTA antibody research in the future?

Several emerging technologies hold promise for advancing ERECTA antibody research:

• Recombinant antibody development: Following approaches like those of the Protein Capture Reagents Program (PCRP) or NeuroMab, developing highly specific recombinant antibodies against ERECTA could provide more consistent and well-characterized reagents .

• Proximity labeling techniques: Methods like BioID or APEX2 fused to ERECTA could identify proteins in close proximity in vivo, with results validated using antibody-based techniques.

• Super-resolution microscopy: Advanced imaging with ERECTA antibodies could reveal nanoscale organization of receptor complexes at the plasma membrane, providing new insights into signaling cluster formation.

• Single-molecule tracking: Combining antibody fragments with quantum dots or other fluorescent tags could enable tracking of individual ERECTA molecules, revealing dynamics not visible in population-based studies.

• Spatial proteomics: New approaches combining antibody-based detection with spatial resolution could map ERECTA distribution across tissues and subcellular locations with unprecedented detail.

• Multiplexed antibody detection: Methods allowing simultaneous detection of multiple proteins could reveal complex relationships between ERECTA and its various signaling partners, co-receptors, and regulators in single samples.

These technologies align with broader trends in improving antibody-based research across the scientific community, as highlighted by initiatives focused on enhancing reagent quality and characterization .