CXE1 Antibody

What is CXE1 protein and what biological role does it play in Arabidopsis thaliana?

CXE1 (Carboxylesterase 1) is a protein expressed in Arabidopsis thaliana with the UniProt accession number Q9LMA7. It belongs to the carboxylesterase family and is involved in hydrolytic activity, playing roles in plant defense mechanisms and metabolic processes. CXE1 contributes to the plant's ability to hydrolyze ester bonds in various substrates, which may be important for detoxification pathways and responses to environmental stresses .

What are the primary applications of CXE1 Antibody in plant research?

CXE1 Antibody is primarily used for detecting and quantifying CXE1 protein expression in Arabidopsis thaliana tissues. Common applications include:

• Western blotting for protein expression analysis

• Immunohistochemistry for localization studies

• Immunoprecipitation for protein-protein interaction studies

• Flow cytometry for quantitative analysis in cell populations

Similar to methodologies established for other plant antibodies, CXE1 Antibody can be used to study protein expression patterns during different developmental stages or in response to environmental stimuli .

What technical specifications should researchers know about commercially available CXE1 Antibody?

Commercial CXE1 Antibody (such as product code CSB-PA873296XA01DOA) typically has the following specifications:

This antibody recognizes the native CXE1 protein from Arabidopsis thaliana and can be used in various experimental contexts requiring specific detection of this protein .

What are the optimal conditions for using CXE1 Antibody in Western blotting?

For optimal Western blot results with CXE1 Antibody, researchers should consider the following protocol:

• Sample preparation: Extract proteins from Arabidopsis tissues using a buffer containing appropriate protease inhibitors to prevent degradation.

• Gel electrophoresis: Separate proteins on a 10-12% SDS-PAGE gel, loading 20-50 μg of total protein per lane.

• Transfer conditions: Transfer to PVDF or nitrocellulose membrane at 100V for 60-90 minutes in cold transfer buffer.

• Blocking: Block with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute CXE1 Antibody at 1:1000 to 1:2000 in blocking buffer, incubate overnight at 4°C.

• Washing: Wash 3-4 times with TBST, 5-10 minutes each.

• Secondary antibody: Incubate with appropriate HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature.

• Detection: Develop using enhanced chemiluminescence (ECL) substrate.

This methodology draws on established protocols for plant antibodies, adapting them specifically for CXE1 detection .

How can researchers validate the specificity of CXE1 Antibody?

To ensure experimental rigor, researchers should validate the specificity of CXE1 Antibody using the following approaches:

• Positive controls: Include recombinant CXE1 protein or extracts from wild-type Arabidopsis known to express CXE1.

• Negative controls: Use protein extracts from cxe1 knockout mutants (if available) or from tissues known not to express CXE1.

• Peptide competition assay: Pre-incubate the antibody with excess purified CXE1 peptide before immunodetection to demonstrate signal specificity.

• Cross-reactivity testing: Test against other related carboxylesterases (e.g., CXE5, CXE13) to ensure specificity within this protein family .

• Multiple detection techniques: Confirm results using at least two different techniques (e.g., Western blot and immunohistochemistry).

This validation approach mirrors established antibody validation methods used for other plant proteins like CAX1, where knockout mutants proved valuable for specificity confirmation .

What optimization strategies are recommended for immunolocalization studies with CXE1 Antibody?

For successful immunolocalization of CXE1 in plant tissues, researchers should consider:

• Fixation method: Use 4% paraformaldehyde for 1-2 hours, followed by dehydration and embedding appropriate for the tissue type.

• Antigen retrieval: Perform heat-induced epitope retrieval in citrate buffer (pH 6.0) if necessary to expose epitopes masked during fixation.

• Section thickness: Prepare 5-10 μm thick sections for optimal antibody penetration.

• Antibody dilution: Begin with 1:100 dilution and optimize based on signal-to-noise ratio.

• Incubation conditions: Incubate sections with primary antibody overnight at 4°C in a humidified chamber.

• Detection system: Use fluorescent secondary antibodies for co-localization studies or HRP-conjugated systems for brightfield microscopy.

• Controls: Include sections without primary antibody and, if possible, tissues from cxe1 mutants as negative controls.

This methodology adapts subcellular localization techniques that have proven successful for other plant proteins like CAX1, which was localized to the tonoplast using similar approaches .

How can CXE1 Antibody be used to investigate protein-protein interactions in planta?

For investigating CXE1 protein-protein interactions, researchers can employ the following methodologies:

• Co-immunoprecipitation (Co-IP):

  • Crosslink proteins in intact plant tissues if transient interactions are expected

  • Prepare cellular lysates under non-denaturing conditions

  • Incubate lysates with CXE1 Antibody bound to protein A/G beads

  • Elute complexes and analyze interacting partners by mass spectrometry

• Proximity Ligation Assay (PLA):

  • Use CXE1 Antibody in conjunction with antibodies against suspected interacting partners

  • Secondary antibodies with attached DNA probes enable visualization of interaction events

  • This provides spatial information about interactions within cellular compartments

• Bimolecular Fluorescence Complementation (BiFC) validation:

  • Use Co-IP results to inform candidate selection for BiFC experiments

  • This combination of antibody-based and genetic approaches provides robust validation

This integrated approach draws on methodologies similar to those used for studying CAX1 protein interactions in Arabidopsis .

What experimental approaches can identify post-translational modifications of CXE1 using the antibody?

To investigate post-translational modifications (PTMs) of CXE1, researchers can implement:

• Phosphorylation analysis:

  • Immunoprecipitate CXE1 using the antibody

  • Perform Western blot with phospho-specific antibodies

  • Alternatively, analyze immunoprecipitated protein by mass spectrometry to identify phosphorylation sites

• Ubiquitination detection:

  • Immunoprecipitate CXE1

  • Probe with anti-ubiquitin antibodies on Western blots

  • Use proteasome inhibitors to enhance detection of ubiquitinated forms

• Glycosylation assessment:

  • Treat immunoprecipitated CXE1 with glycosidases

  • Observe mobility shifts on Western blots with CXE1 Antibody

  • Use lectin blotting as a complementary approach

• Comparative PTM analysis:

  • Apply these techniques to samples from plants under different stress conditions

  • Correlate PTM changes with alterations in CXE1 enzymatic activity

These approaches adapt established methodologies used for studying PTMs in other plant proteins .

How can researchers use CXE1 Antibody to investigate subcellular localization and trafficking?

To study subcellular localization and trafficking of CXE1 protein, researchers should consider:

• Subcellular fractionation with immunoblotting:

  • Perform differential centrifugation to isolate cellular compartments

  • Analyze fractions by Western blotting with CXE1 Antibody

  • Include markers for different organelles (e.g., V-PPase for vacuolar membrane)

  • Present data as distribution profiles across fractionation gradients

• Immunogold electron microscopy:

  • Fix plant tissues with glutaraldehyde and embed in LR White resin

  • Prepare ultrathin sections and incubate with CXE1 Antibody

  • Detect with gold-conjugated secondary antibodies

  • Quantify gold particle distribution across cellular compartments

• Confocal immunofluorescence microscopy:

  • Fix and permeabilize plant tissues

  • Incubate with CXE1 Antibody followed by fluorescent secondary antibody

  • Co-stain with organelle-specific markers

  • Perform co-localization analysis

This approach mirrors successful localization studies for other plant proteins, such as CAX1, which was localized to the tonoplast using gradient fractionation and immunodetection techniques .

What are common technical challenges when working with CXE1 Antibody and how can they be addressed?

Researchers commonly encounter these challenges when working with plant antibodies like CXE1 Antibody:

These troubleshooting strategies are informed by approaches used with other plant antibodies in research settings .

How should researchers design controls when using CXE1 Antibody in complex experimental designs?

For robust experimental design with CXE1 Antibody, implement these controls:

• Technical controls:

  • No primary antibody control to assess secondary antibody specificity

  • Isotype control (irrelevant antibody of same isotype) to evaluate non-specific binding

  • Pre-immune serum control (if available) for polyclonal antibody background assessment

• Biological controls:

  • Wild-type Arabidopsis samples as positive controls

  • cxe1 knockout mutants as negative controls (if available)

  • Dose-response controls (varying amounts of target protein)

  • Recombinant CXE1 protein as reference standard

• Experimental validation:

  • Complementary detection methods (e.g., mass spectrometry)

  • Independent antibody targeting a different epitope of CXE1

  • Cross-validation using genetic approaches (e.g., GFP-tagged CXE1)

This comprehensive control strategy enhances experimental rigor and facilitates troubleshooting, following best practices in antibody-based research on plant proteins .

How can researchers optimize CXE1 Antibody for flow cytometry applications?

To optimize CXE1 Antibody for flow cytometry in plant cell suspensions:

• Sample preparation:

  • Generate protoplasts using appropriate enzymes for cell wall digestion

  • Maintain cell viability with osmotic stabilizers

  • Fix cells with 2-4% paraformaldehyde if intracellular staining is required

• Permeabilization:

  • Use 0.1% Triton X-100 or 0.05% saponin for intracellular targets

  • Optimize concentration and duration to maintain cell integrity

• Antibody titration:

  • Test dilutions ranging from 1:50 to 1:1000

  • Determine optimal concentration based on signal-to-noise ratio

• Staining protocol:

  • Block with 5% BSA in PBS for 30 minutes

  • Incubate with primary antibody for 1 hour at room temperature

  • Wash thoroughly between steps

  • Use fluorophore-conjugated secondary antibody appropriate for flow cytometer configuration

• Controls for gating:

  • Unstained cells for autofluorescence assessment

  • Secondary antibody-only control

  • Isotype control

This optimization strategy draws on methodologies similar to those used for developing the Cx1Mab-1 antibody for flow cytometry, adapted for plant cell applications .

How should researchers quantify and normalize CXE1 protein expression data?

For accurate quantification and normalization of CXE1 protein expression:

• Western blot quantification:

  • Use densitometric analysis with linear dynamic range verification

  • Analyze multiple exposure times to ensure measurements in linear range

  • Create standard curves using recombinant CXE1 protein at known concentrations

• Normalization strategies:

  • Normalize to total protein (measured by Ponceau S or amido black staining)

  • Use multiple housekeeping proteins (e.g., actin, tubulin, GAPDH) rather than a single reference

  • Consider the stability of reference proteins under your experimental conditions

• Statistical analysis:

  • Perform a minimum of three biological replicates

  • Apply appropriate statistical tests based on data distribution

  • Report effect sizes alongside p-values

• Data presentation:

  • Present representative images alongside quantification

  • Include both raw and normalized data

  • Report variability measures (e.g., standard deviation, confidence intervals)

This approach ensures rigorous quantification similar to established methods used in plant protein expression studies .

What considerations are important when interpreting CXE1 localization in different plant tissues and cell types?

When interpreting CXE1 localization data, researchers should consider:

• Tissue-specific expression patterns:

  • Compare CXE1 expression across different tissues (roots, leaves, stems, flowers)

  • Correlate protein localization with known function in specific tissues

  • Consider developmental stage effects on expression patterns

• Cell type heterogeneity:

  • Distinguish between cell types within a tissue sample

  • Compare expression in specialized cells (e.g., guard cells, trichomes)

  • Use cell-type-specific markers for co-localization studies

• Subcellular distribution:

  • Assess distribution across membrane compartments

  • Consider dynamic relocalization in response to stimuli

  • Compare with predicted localization based on sequence analysis

• Technical considerations:

  • Account for fixation artifacts

  • Consider antibody accessibility limitations in different tissues

  • Validate findings with complementary approaches (e.g., fluorescent protein fusions)

This comprehensive interpretation approach draws on established practices in plant cell biology research, similar to the localization studies conducted for CAX1 protein in Arabidopsis .

How can CXE1 Antibody be used in conjunction with genetic approaches to study CXE1 function?

Researchers can combine antibody-based detection with genetic approaches through:

• Knockout/knockdown validation:

  • Use CXE1 Antibody to confirm absence/reduction of protein in cxe1 mutants

  • Quantify residual protein in knockdown lines

  • Compare phenotypes with protein expression levels

• Complementation studies:

  • Verify protein expression in complemented lines

  • Correlate functional rescue with expression levels

  • Assess localization of native vs. transgenic protein

• Structure-function analysis:

  • Use the antibody to detect truncated or mutated versions of CXE1

  • Correlate protein stability with functional outcomes

  • Assess effects of mutations on subcellular localization

• Conditional expression systems:

  • Monitor protein accumulation in inducible expression systems

  • Correlate timing of protein accumulation with phenotypic changes

  • Study protein turnover rates following induction

This integrated approach combines the strengths of antibody-based detection with genetic manipulation, similar to strategies used for studying CAX1 function in Arabidopsis .

What proteomics approaches can be enhanced using CXE1 Antibody?

CXE1 Antibody can enhance proteomic analyses through:

• Immunoprecipitation-mass spectrometry (IP-MS):

  • Enrich CXE1 and interacting proteins using the antibody

  • Identify interaction partners by mass spectrometry

  • Compare interactomes under different conditions or treatments

• Targeted proteomics:

  • Develop selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) assays

  • Use antibody-based enrichment to improve detection of low-abundance CXE1

  • Quantify specific post-translational modifications

• Protein complex analysis:

  • Use antibody for native protein complex isolation

  • Combine with blue native PAGE to preserve complex integrity

  • Identify complex components by mass spectrometry

• Spatial proteomics:

  • Combine immunoprecipitation with subcellular fractionation

  • Map CXE1 distribution across cellular compartments

  • Identify compartment-specific interaction partners

These proteomics approaches leverage the specificity of CXE1 Antibody to enhance detection and characterization beyond standard techniques, following methodologies established for other plant proteins .