CLE40 Antibody

What is CLE40 and why would researchers need antibodies against it?

CLE40 is a secreted peptide belonging to the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) family that controls stem cell homeostasis in plant root meristems. It acts through receptor-like kinases (RLKs) including ARABIDOPSIS CRINKLY4 (ACR4) and CLAVATA1 (CLV1) in the distal root meristem to promote cell differentiation, while in the proximal meristem it works through CLAVATA2 (CLV2) and CORYNE (CRN) to inhibit cell differentiation . Antibodies against CLE40 are essential tools for studying its expression patterns, protein localization, and signaling pathways in plant development research.

What validation strategies are essential when selecting a CLE40 antibody?

When selecting a CLE40 antibody, researchers should employ multiple validation strategies based on the "five pillars" approach:

• Genetic strategy validation: Test antibody specificity using CLE40 knockout or knockdown plant materials

• Orthogonal strategy validation: Compare antibody-based detection with antibody-independent methods like RNA-seq

• Independent antibody validation: Use multiple antibodies targeting different epitopes of CLE40

• Recombinant expression validation: Test with recombinantly expressed CLE40 protein

• Immunocapture MS validation: Verify captured proteins by mass spectrometry

Each experimental context requires specific validation, as antibody performance is highly context-dependent.

How should researchers distinguish between CLE40 and other closely related CLE peptides?

Due to the high sequence similarity among CLE family peptides, researchers must:

• Select antibodies raised against unique regions of CLE40 that differ from other CLE peptides

• Perform cross-reactivity tests against other CLE peptides, particularly CLE14 which is also expressed in root tissues

• Include appropriate controls using tissues with known expression patterns of different CLE peptides

• Consider using epitope-tagged CLE40 constructs if specific antibodies are unavailable

• Validate results using genetic approaches with CLE40 mutants alongside antibody-based detection

What are the recommended protocols for using CLE40 antibodies in immunolocalization studies?

For optimal immunolocalization of CLE40 in plant tissues:

• Tissue preparation:

  • Fix tissues in 4% paraformaldehyde

  • Consider the developmental stage carefully (10 DAG plants show better CLE40 responses than younger plants)

  • Use thin sections (5-8 μm) to enhance antibody penetration

• Antigen retrieval:

  • Perform heat-induced epitope retrieval if necessary

  • Consider cell wall digestion with enzymes for better antibody access

• Blocking and antibody incubation:

  • Block with 3-5% BSA in PBS with 0.1% Triton X-100

  • Incubate with primary CLE40 antibody (1:100-1:500 dilution range)

  • Use secondary antibodies conjugated to fluorescent dyes for visualization

• Controls:

  • Include CLE40 knockout/knockdown tissues as negative controls

  • Pre-adsorb antibody with recombinant CLE40 peptide as specificity control

How can researchers optimize Western blot conditions for CLE40 detection?

Optimizing Western blot conditions for CLE40 detection requires:

• Sample preparation:

  • Extract proteins using buffers containing protease inhibitors

  • Enrich for membrane fractions or secreted proteins depending on experimental goals

  • Consider immunoprecipitation to concentrate low-abundance CLE40

• Gel electrophoresis parameters:

  • Use higher percentage (15-18%) gels for better resolution of small peptides

  • Include positive controls with recombinant CLE40 peptide

  • Consider using Tricine-SDS-PAGE for better separation of low molecular weight proteins

• Transfer and detection optimization:

  • Use PVDF membranes (0.2 μm pore size) for small peptides

  • Optimize transfer conditions (shorter time, lower voltage)

  • Use enhanced chemiluminescence (ECL) or near-infrared detection systems

• Validation controls:

  • Run samples from CLE40 knockout plants alongside wild-type

  • Include recombinant CLE40 at known concentrations as standard curve

What methodological approaches are recommended for co-immunoprecipitation experiments involving CLE40?

For co-immunoprecipitation of CLE40 and its interacting partners:

• Cross-linking approach:

  • Consider in vivo cross-linking with DSP or formaldehyde to stabilize transient interactions

  • Optimize cross-linking time to capture signaling complexes

• Lysis conditions:

  • Use mild detergents (0.5-1% NP-40 or digitonin) to preserve protein-protein interactions

  • Include phosphatase inhibitors to maintain phosphorylation states critical for receptor interactions

• Immunoprecipitation strategy:

  • Pre-clear lysates to reduce non-specific binding

  • Use antibodies against CLE40 receptors (ACR4, CLV1, CLV2, CRN) to pull down complexes

  • Consider epitope-tagged versions of CLE40 for more efficient pull-down

• Detection methods:

  • Western blot for known interactors

  • Mass spectrometry for unbiased identification of novel interacting partners

How can researchers address non-specific binding issues with CLE40 antibodies?

To address non-specific binding:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, milk, normal serum)

  • Increase blocking time or concentration

  • Add 0.1-0.3% Tween-20 to reduce hydrophobic interactions

• Antibody titration:

  • Perform careful dilution series to determine optimal concentration

  • Consider using antibody purification techniques if necessary

• Absorption controls:

  • Pre-incubate antibody with recombinant CLE40 peptide to compete away specific binding

  • Compare staining patterns with and without absorption

• Alternative validation approaches:

  • Use orthogonal detection methods when possible

  • Compare results with genetic controls (CLE40 knockout or overexpression)

What statistical methods are appropriate for quantifying CLE40 expression differences between experimental groups?

For robust quantification:

• Image analysis parameters:

  • Use consistent acquisition settings between samples

  • Perform background subtraction using appropriate controls

  • Define regions of interest (ROIs) consistently between samples

• Normalization approaches:

  • Normalize to internal standards or housekeeping proteins

  • Consider using ratios to reference proteins rather than absolute values

  • Account for tissue-specific expression patterns

• Statistical tests:

  • For normally distributed data: t-tests (two groups) or ANOVA (multiple groups)

  • For non-parametric data: Mann-Whitney U or Kruskal-Wallis tests

  • Include post-hoc tests for multiple comparisons (Tukey's, Bonferroni)

• Data presentation:

  • Present both representative images and quantitative analyses

  • Include appropriate error bars (SD for data distribution, SEM for precision of mean)

  • Report biological replicates (n) and technical replicates

How can researchers distinguish between specific CLE40 signal and background in immunofluorescence studies?

To distinguish specific signal from background:

• Multiple control approaches:

  • No primary antibody controls

  • CLE40 knockout tissue controls

  • Peptide competition assays

  • Secondary antibody-only controls

• Signal validation techniques:

  • Compare antibody staining pattern with known CLE40 mRNA expression

  • Evaluate signal consistency across biological replicates

  • Examine signal localization (CLE40 should be detectable in differentiated columella cells and stele)

• Advanced imaging approaches:

  • Use spectral unmixing to separate signal from autofluorescence

  • Implement deconvolution algorithms to improve signal-to-noise ratio

  • Consider super-resolution techniques for detailed subcellular localization

How can CLE40 antibodies be utilized to study calcium signaling mechanisms triggered by CLE40?

CLE40 peptide triggers a rapid but spatially localized increase in cytosolic calcium concentration in root meristem cells . To study this mechanism:

• Combined calcium imaging and immunolabeling:

  • Use GCaMP6-expressing plants to visualize calcium fluxes

  • Follow with CLE40 immunolabeling to correlate CLE40 localization with calcium response regions

  • Establish temporal relationships between CLE40 binding and calcium flux

• Receptor co-localization studies:

  • Perform triple labeling with antibodies against:

    • CLE40

    • Calcium channels (CNGC9)

    • Receptor complexes (CLV1/BAM1)

  • Analyze subcellular co-localization patterns before and after CLE40 treatment

• Quantitative analysis of calcium response:

  • Measure calcium elevation in specific regions of interest (ROIs)

  • Compare response in wild-type vs. receptor mutants (clv1, bam1, clv2/crn)

  • Create temporal profiles of calcium response following CLE40 application

What advanced techniques can be used to investigate CLE40-receptor interactions using antibodies?

For studying CLE40-receptor interactions:

• Proximity ligation assay (PLA):

  • Use antibodies against both CLE40 and its receptors (ACR4, CLV1)

  • Visualize protein-protein interactions with single-molecule resolution

  • Quantify interaction events in different cell types or conditions

• FRET-based approaches:

  • Label CLE40 antibodies and receptor antibodies with FRET-compatible fluorophores

  • Measure energy transfer to detect close proximity

  • Analyze temporal dynamics of interactions

• Immunoelectron microscopy:

  • Use gold-labeled antibodies against CLE40 and receptors

  • Visualize precise subcellular localization at nanometer resolution

  • Examine membrane domains and endocytic compartments involved in signaling

• Receptor activation studies:

  • Develop phospho-specific antibodies against activated receptors

  • Monitor receptor phosphorylation state following CLE40 treatment

  • Track temporal activation patterns in different cell types

How might CLE40 antibodies be integrated with functional genomics approaches to understand downstream signaling pathways?

Integration of CLE40 antibodies with functional genomics:

• ChIP-seq applications:

  • Use antibodies against transcription factors regulated by CLE40 signaling

  • Identify genomic binding sites altered by CLE40 treatment

  • Create regulatory networks downstream of CLE40

• Phosphoproteomics integration:

  • Combine CLE40 immunoprecipitation with phosphoproteomic analysis

  • Identify phosphorylation changes triggered by CLE40 signaling

  • Map kinase cascades responding to CLE40

• Spatial transcriptomics correlation:

  • Perform CLE40 immunolabeling on tissue sections

  • Correlate with spatial transcriptomics data

  • Identify gene expression changes in cells with active CLE40 signaling

• Multi-omics data integration:

  • Create computational models incorporating:

    • CLE40 localization data from antibody studies

    • Calcium imaging data

    • Transcriptomic responses

    • Protein interaction networks

  • Develop predictive models of CLE40 signaling outcomes