CSLA11 Antibody

What is CSLA11 and why is it important in plant research?

CSLA11 (Cellulose Synthase-Like A11) belongs to the glycosyltransferase family and functions as a probable mannan synthase exhibiting 4-β-mannosyltransferase activity. It plays a critical role in plant cell wall biosynthesis and structure. Antibodies targeting this protein are essential for studying plant cell wall development, particularly in model organisms like Arabidopsis thaliana (AT5G16190). Methodologically, researchers utilize CSLA11 antibodies to investigate mannan polymer synthesis and deposition patterns across different plant tissues, developmental stages, and stress responses.

How does CSLA11 Antibody differ from other CSL family antibodies?

CSLA11 Antibody specifically targets the CSLA11 protein, whereas other antibodies like CSL311 target entirely different proteins such as the β common receptor involved in cytokine signaling pathways in animals . Similarly, CSHL1/CSL antibodies recognize chorionic somatomammotropin hormone-like proteins in mammals . When designing experiments, researchers must carefully validate antibody specificity using Western blot analysis against recombinant CSLA proteins to ensure selective binding to CSLA11 and not to other CSLA family members (CSLA1-9) that share sequence homology but may have distinct functional roles in plant cell wall synthesis.

What are the recommended applications for CSLA11 Antibody?

CSLA11 Antibody can be employed in multiple experimental approaches:

• Western blotting (1:500-1:1000 dilution) for protein expression analysis

• Immunohistochemistry (1:100-1:200) for tissue localization studies

• Immunoprecipitation for protein-protein interaction studies

• ChIP assays for transcriptional regulation studies

For quantitative analysis of CSLA11 expression levels, Western blot analysis with appropriate loading controls (e.g., actin or tubulin) is recommended, while immunohistochemistry provides valuable insights into tissue-specific localization patterns.

What controls should be included when using CSLA11 Antibody in immunoblotting experiments?

When designing immunoblotting experiments with CSLA11 Antibody, the following controls are essential:

• Positive control: Protein extracts from tissues known to express CSLA11 (e.g., developing seedlings)

• Negative control: Extracts from csla11 knockout mutants or tissues with negligible CSLA11 expression

• Blocking peptide control: Pre-incubation of antibody with the immunizing peptide to verify specificity

• Loading control: Antibodies against constitutively expressed proteins (e.g., actin, GAPDH)

• Cross-reactivity control: Testing against recombinant proteins of other CSLA family members

This comprehensive control strategy helps distinguish specific signals from background noise and validates antibody specificity, particularly important when working with members of large protein families with high sequence homology.

How should samples be prepared for optimal CSLA11 detection?

For optimal CSLA11 detection:

• Extract total protein from plant tissues using a buffer containing:

  • 50 mM Tris-HCl (pH 8.0)

  • 150 mM NaCl

  • 1% Triton X-100

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail

• Include 1% SDS in the extraction buffer to solubilize membrane-associated proteins

• Perform tissue homogenization at 4°C to prevent protein degradation

• Centrifuge at 12,000 × g for 15 minutes to remove cell debris

• Quantify protein concentration using Bradford or BCA assay

• Use fresh samples when possible, as freeze-thaw cycles can reduce antigen recognition

This methodology maximizes protein extraction while preserving antibody epitopes for detection, critical for membrane-associated glycosyltransferases like CSLA11.

What are the recommended parameters for immunohistochemical detection of CSLA11?

For effective immunohistochemical detection of CSLA11:

• Fix plant tissues in 4% paraformaldehyde for 12-24 hours

• Embed in paraffin or freeze in OCT compound

• Section tissues at 5-10 μm thickness

• Perform antigen retrieval using sodium citrate buffer (pH 6.0) at 95°C for 20 minutes

• Block with 5% normal goat serum in PBS-T for 1 hour

• Incubate with CSLA11 Antibody at 1:100 dilution overnight at 4°C

• Apply secondary antibody conjugated with fluorophore or HRP

• Counterstain with DAPI to visualize nuclei

• Include csla11 knockout tissues as negative controls

These parameters optimize signal-to-noise ratio while preserving tissue morphology for accurate localization studies. When studying developing plant tissues, developmental stage-matched controls are crucial for comparative analyses.

How can cross-reactivity with other CSLA family members be assessed and minimized?

To assess and minimize cross-reactivity:

• Sequence alignment analysis: Compare the immunogenic peptide sequence against all CSLA family members to identify potential cross-reactive epitopes

• Recombinant protein panel testing: Express and purify all CSLA family members and perform Western blot analysis to determine binding specificity

• Knockout validation: Test antibody against tissues from csla11 knockout plants - any remaining signal indicates cross-reactivity

• Peptide competition assay: Pre-incubate antibody with excess immunogenic peptide before application to eliminate specific binding

• Immunoprecipitation-mass spectrometry: Identify all proteins pulled down by the antibody to detect unintended targets

This systematic approach allows researchers to confidently interpret experimental results by quantifying potential cross-reactivity with homologous proteins.

What are common causes of inconsistent results when using CSLA11 Antibody?

Inconsistent results may arise from:

• Antibody batch variation: Different lots may have varying affinities; always validate new batches against a reference sample

• Sample preparation inconsistencies: Variations in extraction methods, buffer compositions, or protein degradation during processing

• Environmental factors affecting target expression: Light conditions, growth stage, and stress exposure can dramatically alter CSLA11 expression patterns

• Technical variations: Inconsistent transfer efficiency, incubation times, or detection methods

• Storage conditions: Antibody activity may decrease with repeated freeze-thaw cycles or extended storage at suboptimal temperatures

To minimize variability, researchers should standardize protocols, include reference samples across experiments, and document all experimental conditions meticulously.

How can the specificity of CSLA11 Antibody be validated in transformant research?

To validate antibody specificity in transformant research:

• Overexpression systems: Generate plants overexpressing CSLA11 with epitope tags (e.g., HA, FLAG) and confirm co-detection with both tag-specific and CSLA11-specific antibodies

• Inducible expression systems: Create lines with inducible CSLA11 expression and confirm correlation between induction level and antibody signal intensity

• RNA interference (RNAi): Develop CSLA11 knockdown lines and verify reduced antibody signal corresponding to reduced transcript levels

• CRISPR/Cas9 knockout validation: Generate precise gene knockouts and confirm complete absence of antibody signal

• Heterologous expression: Express CSLA11 in non-plant systems (e.g., yeast, bacteria) and verify antibody detection in these systems

This multi-faceted validation approach provides compelling evidence for antibody specificity across diverse experimental contexts.

How can CSLA11 Antibody be utilized in studying protein-protein interactions within cell wall biosynthesis complexes?

For studying protein-protein interactions:

• Co-immunoprecipitation (Co-IP): Use CSLA11 Antibody to pull down protein complexes, followed by mass spectrometry to identify interaction partners

• Proximity labeling: Combine with BioID or APEX2 techniques by creating fusion proteins that biotinylate proximal proteins, then use CSLA11 Antibody to verify proper fusion protein localization

• Förster Resonance Energy Transfer (FRET): Use fluorescently-labeled CSLA11 Antibody in combination with antibodies against potential interaction partners to detect molecular proximity (<10 nm)

• Bimolecular Fluorescence Complementation (BiFC): Validate interactions identified by antibody-based methods using split fluorescent protein complementation assays

• Sucrose density gradient fractionation: Use CSLA11 Antibody to track co-sedimentation with other cell wall biosynthesis proteins across gradient fractions

These methodologies provide complementary approaches to map the interaction network of CSLA11 within the cell wall biosynthesis machinery.

What advanced imaging techniques can be combined with CSLA11 Antibody for subcellular localization studies?

Advanced imaging approaches include:

• Super-resolution microscopy: Techniques like STED, PALM, or STORM can resolve CSLA11 localization beyond the diffraction limit (approximately 200 nm), revealing distinct subcellular compartments and potential microdomains

• Correlative Light and Electron Microscopy (CLEM): Combine immunofluorescence using CSLA11 Antibody with electron microscopy to correlate protein localization with ultrastructural features

• Expansion microscopy: Physically expand the sample using swellable polymers to achieve super-resolution imaging with standard confocal microscopes

• Live-cell imaging: Pair with genetically encoded fluorescent proteins to track dynamic movements of CSLA11-containing complexes in real time

• Multi-color 3D confocal microscopy: Co-localize CSLA11 with organelle markers to establish precise subcellular distribution patterns

These techniques provide unprecedented spatial resolution for understanding CSLA11 trafficking and localization in relation to cell wall deposition sites.

How can CSLA11 Antibody be employed in comparative studies across different plant species?

For cross-species comparative studies:

• Epitope conservation analysis: Perform sequence alignment of the antibody epitope region across species to predict cross-reactivity

• Western blot validation: Test antibody recognition across protein extracts from multiple species with varying phylogenetic distances from Arabidopsis

• Dot blot titration: Determine minimum detection thresholds and relative affinities across species by applying serial dilutions of protein extracts

• Immunohistochemistry optimization: Adjust fixation and antigen retrieval protocols for different species based on tissue composition and density

• Cross-linking efficiency testing: If using cross-linking reagents, optimize conditions for each species due to variations in cell wall composition

This systematic approach enables reliable comparative studies of CSLA11 across diverse plant lineages, providing insights into evolutionary conservation of mannan synthesis mechanisms.

How might CSLA11 Antibody contribute to studies of plant response to environmental stress?

CSLA11 Antibody can illuminate stress response mechanisms through:

• Stress-induced expression profiling: Quantify CSLA11 protein levels across various abiotic stresses (drought, salinity, temperature extremes) using immunoblotting

• Spatial reorganization analysis: Employ immunohistochemistry to track changes in CSLA11 localization patterns during stress adaptation

• Post-translational modification detection: Develop modification-specific antibodies to detect stress-induced phosphorylation, glycosylation, or other modifications of CSLA11

• Protein stability assessment: Measure CSLA11 protein half-life under stress conditions using cycloheximide chase experiments followed by immunoblotting

• Cell wall composition correlation: Combine antibody-based CSLA11 quantification with comprehensive cell wall polymer analysis to establish structure-function relationships during stress adaptation

These approaches can reveal how plants modulate cell wall biosynthesis to enhance stress resilience, potentially informing strategies for developing climate-resilient crops.

What role can CSLA11 Antibody play in advancing our understanding of evolutionary patterns in plant cell wall biosynthesis?

CSLA11 Antibody enables evolutionary insights through:

• Phylogenetic expression mapping: Quantify CSLA11 protein levels across diverse plant lineages to identify evolutionary shifts in expression patterns

• Functional domain conservation analysis: Use epitope-specific antibodies targeting different protein domains to assess evolutionary conservation of functional regions

• Developmental regulation comparison: Track ontogenetic changes in CSLA11 expression across plant lineages to identify conserved vs. divergent developmental programs

• Associated protein network evolution: Perform comparative co-immunoprecipitation studies across species to map evolutionary changes in protein-protein interaction networks

• Structure-function relationship studies: Correlate antibody-detected CSLA11 expression with species-specific variations in mannan content and structure

This evolutionary perspective provides a deeper understanding of how plant cell wall biosynthesis machinery has adapted across plant diversification, potentially revealing novel biotechnological targets.

How can CSLA11 Antibody be integrated with multi-omics approaches for comprehensive cell wall studies?

Integration with multi-omics approaches involves:

• Proteogenomic correlation: Combine CSLA11 protein quantification via immunoblotting with transcriptomic data to identify post-transcriptional regulation mechanisms

• Metabolomic integration: Correlate CSLA11 protein levels with metabolite profiles of cell wall precursors to establish metabolic flux relationships

• Glycomic association: Link CSLA11 expression patterns detected by immunohistochemistry with comprehensive cell wall polysaccharide compositional analysis

• Phenomic correlation: Associate CSLA11 protein levels with high-throughput phenotyping data to establish genotype-protein-phenotype relationships

• Systems biology modeling: Incorporate quantitative CSLA11 protein data into predictive models of cell wall biosynthesis and remodeling

This integrated approach provides a systems-level understanding of how CSLA11 functions within the broader context of plant cell wall biosynthesis and development.