CLE25 Antibody

What is CLE25 and why is it significant in plant research?

CLE25 is a CLAVATA3/ESR (CLE)-related protein that plays a crucial role in plant stress responses. Its significance lies in its function as a long-distance signaling molecule during dehydration stress. CLE25 produces a graft-transmissible signal that travels from root to shoot, inducing abscisic acid (ABA) synthesis and resulting in stomatal closure, which helps plants conserve water during drought conditions . This signaling pathway represents an important adaptation mechanism in plants, making CLE25 a valuable target for researchers studying plant responses to environmental stresses.

What species cross-reactivity does the CLE25 antibody demonstrate?

The CLE25 antibody shows specific cross-reactivity with several plant species in the Brassicaceae family. According to product specifications, the antibody effectively recognizes CLE25 in Arabidopsis thaliana (the model plant organism), as well as agriculturally important species like Brassica napus (rapeseed/canola) and Brassica rapa (field mustard) . This cross-reactivity makes the antibody valuable for comparative studies across related plant species, allowing researchers to investigate evolutionary conservation of CLE25 signaling mechanisms.

How should CLE25 antibody be stored and handled to maintain efficacy?

For optimal results with CLE25 antibody, proper storage and handling protocols are essential:

• Storage condition: The antibody is typically supplied in lyophilized form and should be stored in a manual defrost freezer at recommended temperatures .

• Reconstitution: Follow manufacturer's protocols for proper reconstitution before use.

• Avoid freeze-thaw cycles: Repeated freeze-thaw cycles can damage antibody structure and reduce efficacy .

• Shipping condition: The product is shipped at 4°C and should be stored immediately at the recommended temperature upon receipt .

• Working aliquots: For frequent use, consider preparing small working aliquots to minimize freeze-thaw cycles.

What are the common applications for CLE25 antibody in plant research?

CLE25 antibody can be utilized in several experimental techniques:

• Western blotting: For detecting and quantifying CLE25 protein expression levels in plant tissues.

• Immunohistochemistry/Immunofluorescence: For visualizing the spatial distribution of CLE25 in plant tissues and determining its cellular and subcellular localization.

• Immunoprecipitation: For isolating CLE25 and its associated protein complexes.

• ELISA: For quantitative measurement of CLE25 levels in plant extracts.

• Flow cytometry: Though less common in plant research, this technique can be adapted for analyzing CLE25 in plant protoplasts or isolated cells.

These applications enable researchers to investigate CLE25's role in drought response signaling pathways and other potential functions in plant physiology.

How can I optimize Western blot protocols for CLE25 detection?

For optimal Western blot detection of CLE25, consider the following technical adjustments:

• Sample preparation: Use a plant protein extraction buffer containing protease inhibitors to prevent degradation of CLE25.

• Protein separation: Use 12-15% SDS-PAGE gels for optimal separation of CLE25, which is a small peptide.

• Transfer optimization: Use PVDF membranes with 0.2 μm pore size rather than 0.45 μm to better retain small peptides.

• Blocking optimization: Test both BSA and non-fat dry milk as blocking agents to determine which provides better signal-to-noise ratio.

• Antibody dilution: Start with manufacturer's recommended dilution (typically 1:1000) but optimize based on signal strength and background.

• Enhanced detection: Consider using enhanced chemiluminescence (ECL) substrates with higher sensitivity for detection of low-abundance CLE25.

• Controls: Always include positive controls (purified CLE25 or extracts from tissues known to express high levels of CLE25) and negative controls (tissues with low or no CLE25 expression).

What approaches can be used to validate CLE25 antibody specificity?

Validating antibody specificity is crucial for reliable experimental results. For CLE25 antibody, consider these validation methods:

• Peptide competition assay: Pre-incubate the antibody with purified CLE25 peptide before application to samples. Signal reduction indicates specific binding.

• Genetic controls: Compare signal between wild-type plants and cle25 knockout/knockdown mutants or plants overexpressing CLE25.

• Multiple antibody comparison: If available, use different CLE25 antibodies targeting distinct epitopes and compare detection patterns.

• Cross-species validation: Test antibody reactivity in species with known sequence homology to confirm expected cross-reactivity pattern .

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the precipitated protein.

• Recombinant protein controls: Use purified recombinant CLE25 protein as a positive control for size verification.

A comprehensive validation approach strengthens confidence in experimental results and helps identify potential cross-reactivity issues.

How can I determine the optimal fixation method for immunohistochemistry with CLE25 antibody?

Fixation is critical for preserving tissue architecture while maintaining antibody epitope accessibility. For CLE25 immunohistochemistry:

• Compare fixatives: Test both cross-linking fixatives (4% paraformaldehyde) and precipitating fixatives (acetone, methanol) to determine which best preserves CLE25 epitopes.

• Fixation timing: Optimize fixation duration - too short leads to inadequate fixation, while too long may mask epitopes.

• Antigen retrieval methods: If signal is weak after fixation, evaluate heat-induced epitope retrieval (HIER) or enzymatic retrieval methods.

• Buffer composition: Test different fixation buffers (phosphate, citrate) at various pH values (6.0-8.0) to optimize epitope preservation.

• Fixation temperature: Compare room temperature versus 4°C fixation to determine optimal conditions.

• Tissue thickness: Adjust section thickness (5-10 μm for paraffin sections, 40-100 μm for vibratome sections) to optimize antibody penetration.

• Permeabilization: For subcellular localization, optimize permeabilization with detergents (0.1-0.5% Triton X-100) to improve antibody access.

Systematic comparison of these parameters will help establish robust immunohistochemistry protocols for CLE25 detection.

How can I use CLE25 antibody to investigate drought-responsive signaling cascades?

Investigating CLE25's role in drought response requires sophisticated experimental approaches:

• Tissue-specific expression analysis: Use CLE25 antibody for immunohistochemistry to map expression patterns in different tissues before and during drought stress. Focus on vascular tissues where long-distance signaling occurs.

• Co-immunoprecipitation studies: Use CLE25 antibody to pull down protein complexes, then identify interacting partners through mass spectrometry to elucidate signaling components.

• Receptor identification: Combine CLE25 antibody with antibodies against potential receptors (BAM1/BAM3) for co-localization studies to confirm receptor-ligand interactions .

• Phosphorylation cascade analysis: After CLE25 treatment, use phospho-specific antibodies to track activation of downstream signaling components.

• Split-root experiments: Apply CLE25 antibody in neutralization experiments within split-root systems to block signaling in specific root portions and analyze effects on ABA synthesis and stomatal closure.

• Time-course analysis: Use the antibody to track CLE25 protein levels at different time points after drought stress imposition to establish the temporal dynamics of the signaling response.

This multi-faceted approach will help decode the complete signaling pathway from drought perception to stomatal response.

What considerations should be made when designing peptide mapping experiments for CLE25 antibody validation?

Peptide mapping is crucial for antibody validation, especially for complex proteins like CLE25:

• Enzymatic digestion selection: Choose appropriate enzymes (trypsin, chymotrypsin, or Lys-C) based on CLE25's amino acid sequence to generate optimal peptide fragments for analysis .

• Sample preparation: Ensure high purity of CLE25 protein samples to avoid contamination that could complicate interpretation.

• LC/MS optimization: Use high-resolution liquid chromatography coupled with mass spectrometry (LC/MS) systems for accurate peptide identification .

• Epitope mapping: Generate overlapping peptide fragments covering the entire CLE25 sequence to identify which regions are recognized by the antibody.

• Post-translational modification analysis: Assess whether modifications affect antibody recognition by comparing modified and unmodified peptide detection .

• Comparative analysis: Compare peptide maps from different batches of antibody to ensure consistency in epitope recognition .

• Data analysis algorithms: Employ specialized software like MassHunter Qualitative Analysis or similar tools for accurate peptide identification .

Thorough peptide mapping provides critical information about antibody specificity and potential cross-reactivity with similar plant peptides.

How can CLE25 antibody be used to investigate the relationship between CLE25 and its potential receptors BAM1 and BAM3?

Investigating CLE25-receptor interactions requires sophisticated experimental approaches:

• Co-immunoprecipitation: Use CLE25 antibody to pull down protein complexes, then probe with BAM1/BAM3 antibodies to detect interaction. Alternatively, perform reverse co-IP using BAM1/BAM3 antibodies.

• Proximity ligation assay (PLA): Apply CLE25 antibody together with BAM1/BAM3 antibodies to detect in situ protein-protein interactions with subcellular resolution.

• Bimolecular fluorescence complementation (BiFC): While not directly using the antibody, this technique complements antibody-based approaches to visualize CLE25-receptor interactions in living cells.

• Competition assays: Use purified CLE25 peptide to compete with antibody binding and monitor effects on receptor activation or downstream signaling.

• Receptor phosphorylation: After CLE25 treatment, use phospho-specific antibodies to detect BAM1/BAM3 activation.

• Mutant analysis: Compare CLE25-receptor interactions in wild-type plants versus plants with modified CLE25 binding domains in BAM1/BAM3 receptors.

• Super-resolution microscopy: Combine CLE25 and receptor antibodies with techniques like STORM or PALM to visualize nanoscale spatial relationships between CLE25 and its receptors.

These approaches will help establish the molecular mechanisms by which CLE25 interacts with its receptors to initiate drought response signaling .

What are common issues in CLE25 antibody experiments and how can they be resolved?

When working with CLE25 antibody, researchers may encounter several technical challenges:

Systematic troubleshooting using this framework can help resolve most technical issues encountered with CLE25 antibody applications.

How can I adapt flow cytometry protocols for plant cells when using CLE25 antibody?

Flow cytometry with plant cells presents unique challenges that require specific adaptations:

• Protoplast preparation: Develop gentle enzymatic digestion protocols to remove cell walls while preserving CLE25 epitopes.

• Fixation optimization: Compare formaldehyde fixation (2-4%) with alcohol-based fixatives to determine which best preserves CLE25 while enabling antibody access.

• Permeabilization: Test different detergents (Triton X-100, saponin) at various concentrations to optimize intracellular antibody access while minimizing damage to membranes.

• Autofluorescence management: Implement compensation strategies to account for chlorophyll and other plant pigment autofluorescence that may interfere with antibody detection .

• Controls design: Include FMO (Fluorescence Minus One) controls as described for other antibodies to accurately set gates and compensation .

• Antibody titration: Determine optimal antibody concentration specifically for flow cytometry applications, which may differ from other applications.

• Dead cell discrimination: Incorporate viability dyes compatible with plant cells to exclude dead or damaged cells from analysis.

• Data analysis: Apply appropriate gating strategies that account for the unique properties of plant cells and potential autofluorescence issues.

These adaptations will help overcome the specific challenges of applying flow cytometry to plant cells when studying CLE25 expression and localization.

How can I quantitatively assess CLE25 expression levels across different experimental conditions?

For quantitative assessment of CLE25 expression across experimental conditions:

• Western blot quantification:

  • Use internal loading controls (e.g., actin, tubulin, or GAPDH)

  • Apply densitometry analysis with appropriate software (ImageJ, Image Lab)

  • Generate standard curves with purified recombinant CLE25 for absolute quantification

• ELISA development:

  • Optimize sandwich ELISA using CLE25 antibody as capture or detection antibody

  • Develop standard curves with recombinant CLE25 protein

  • Validate assay linearity, sensitivity, and reproducibility across multiple plant samples

• Flow cytometry:

  • Measure mean fluorescence intensity (MFI) as a relative measure of CLE25 expression

  • Use quantitative beads to convert MFI to molecules of equivalent soluble fluorochrome (MESF)

  • Include appropriate controls for each experiment

• qPCR correlation:

  • Perform parallel protein (via antibody) and mRNA quantification

  • Analyze correlation between transcript and protein levels

  • Identify potential post-transcriptional regulation mechanisms

• Microscopy-based quantification:

  • Use confocal microscopy with consistent imaging parameters

  • Apply fluorescence intensity measurement tools

  • Implement automated image analysis for unbiased quantification

These complementary approaches provide robust quantitative assessment of CLE25 expression changes in response to experimental treatments.

How might CLE25 antibody be used to explore potential novel functions of CLE25 beyond drought response?

Beyond its established role in drought response, CLE25 antibody can facilitate exploration of potential novel functions:

• Developmental regulation: Use immunohistochemistry with CLE25 antibody to map expression patterns during different developmental stages to identify potential roles in growth and organogenesis.

• Pathogen response: Investigate CLE25 expression changes during pathogen infection using quantitative approaches with the antibody to uncover potential roles in biotic stress responses.

• Hormone crosstalk: Combine CLE25 antibody-based expression analysis with hormone treatments to identify interactions between CLE25 signaling and plant hormone pathways beyond ABA.

• Root architecture modulation: Given that other CLE peptides regulate root development, use the antibody to study CLE25 distribution in root tissues under various environmental conditions.

• Reproductive development: Examine CLE25 expression in reproductive tissues using the antibody to identify potential roles in flowering, pollination, or seed development.

• Organelle targeting: Use subcellular fractionation followed by Western blotting with CLE25 antibody to identify potential non-canonical localizations that might suggest novel functions.

• Protein-protein interaction networks: Perform large-scale co-immunoprecipitation with CLE25 antibody followed by mass spectrometry to identify novel interacting partners suggesting unexplored functions .

This systematic exploration using CLE25 antibody could reveal multifunctional aspects of this signaling peptide beyond its characterized role in drought response .

What experimental approaches might utilize CLE25 antibody to investigate evolutionary conservation of CLE peptide function across plant species?

Investigating evolutionary conservation of CLE peptide function can be advanced through several antibody-based approaches:

• Cross-species immunodetection: Leverage the documented cross-reactivity with Arabidopsis, Brassica napus, and Brassica rapa to systematically test CLE25 detection in more distantly related plant species.

• Epitope conservation analysis: Map the exact epitope recognized by the antibody and analyze its conservation across plant lineages through sequence alignment and structural prediction.

• Functional complementation with antibody validation: Express CLE25 orthologs from different species in Arabidopsis cle25 mutants, then use the antibody to confirm expression and localization of the heterologous proteins.

• Comparative tissue patterning: Use immunohistochemistry with CLE25 antibody across diverse plant species to compare expression patterns in homologous tissues.

• Receptor binding conservation: Combine CLE25 antibody with receptor antibodies to investigate whether CLE25-receptor interactions are conserved across species.

• Differential post-translational modifications: Use the antibody in combination with mass spectrometry to compare CLE25 post-translational modifications across species that might influence function.

• Ecological adaptation correlation: Compare CLE25 expression patterns using the antibody across species from diverse ecological niches to identify potential adaptive variations in CLE25 signaling.

This evolutionary approach would provide insights into the conservation and diversification of CLE peptide signaling across plant lineages.