CXE3 Antibody

What is CXE3 protein and what biological function does it serve?

CXE3 (Carboxylesterase 3) is a protein encoded by the AT1G49640 gene in Arabidopsis thaliana, commonly known as mouse-ear cress . The protein functions as a carboxylesterase, which is an enzyme that hydrolyzes carboxylic acid esters in plant cells. CXE3 is involved in plant metabolism, particularly in lipid metabolism and detoxification processes. It plays roles in defense mechanisms against xenobiotics and in developmental processes. The protein has a UniProt Number Q9FX92 and is part of the broader carboxylesterase family in plants .

What detection techniques are validated for CXE3 antibody?

The CXE3 antibody (CSB-PA872335XA01DOA) has been validated for enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) applications . These techniques allow for the detection and quantification of CXE3 protein in plant tissue samples. The antibody is provided as an unconjugated preparation, purified using Protein A/G, and is derived from rabbit hosts, making it compatible with standard secondary antibody detection systems used in immunological techniques .

What are the recommended storage conditions for maintaining antibody efficacy?

For optimal preservation of activity, the CXE3 antibody should be stored at either -20°C or -80°C according to manufacturer specifications . Proper aliquoting upon first use is recommended to avoid repeated freeze-thaw cycles that can degrade antibody performance. When shipping is required, the antibody should be transported on blue ice to maintain its cold chain integrity . After thawing for use, store working aliquots at 4°C and use within 1-2 weeks for best results.

What controls should be included when using CXE3 antibody?

When using the CXE3 antibody, researchers should include both positive and negative controls to validate their results. The antibody kit provides 200μg of recombinant immunogen protein/peptide as a positive control . Additionally, 1ml of pre-immune serum is included as a negative control to help distinguish between specific and non-specific binding . For Western blotting applications, researchers should consider running samples from CXE3 knockout plants (if available) or tissues known not to express CXE3 as additional negative controls.

How can Western blot protocols be optimized for CXE3 detection?

For optimal Western blot detection of CXE3 protein in Arabidopsis samples, consider the following protocol adjustments:

• Sample preparation: Use a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, and protease inhibitor cocktail.

• Protein loading: 20-30μg of total protein per lane is typically sufficient.

• Transfer conditions: For this 35-40kDa protein, semi-dry transfer (15V for 30 minutes) or wet transfer (30V overnight at 4°C) both work well.

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

• Primary antibody: Dilute CXE3 antibody 1:1000 to 1:2000 in blocking buffer and incubate overnight at 4°C.

• Secondary antibody: Anti-rabbit IgG HRP-conjugated at 1:5000 dilution for 1 hour at room temperature.

• Detection: Use enhanced chemiluminescence (ECL) substrate with exposure times starting at 1 minute.

This protocol may require adjustment based on protein expression levels and sample types being analyzed.

What are the potential cross-reactivity issues with CXE3 antibody?

The CXE3 antibody is a polyclonal preparation generated against recombinant Arabidopsis thaliana CXE3 protein . Due to its polyclonal nature, it may recognize multiple epitopes on the target protein, which increases sensitivity but may also introduce cross-reactivity with related carboxylesterases in the CXE family. Researchers working with closely related species or investigating multiple CXE proteins should perform preliminary specificity tests. Western blot analysis of recombinant CXE proteins or extracts from plants overexpressing different CXE family members can help determine cross-reactivity profiles.

How do post-translational modifications affect CXE3 antibody binding?

Post-translational modifications (PTMs) of CXE3 can significantly impact antibody recognition efficiency. The CXE3 protein may undergo glycosylation, phosphorylation, or other modifications depending on physiological conditions and developmental stages. If PTMs occur within antibody recognition epitopes, detection efficiency may be reduced. To account for this:

• Use denaturing conditions in Western blotting to potentially expose cryptic epitopes

• Consider treatment with specific enzymes (phosphatases, glycosidases) to remove PTMs before immunodetection

• Compare results across multiple experimental conditions that might influence PTM status

• When working with native protein conformations (as in ELISA), be aware that certain PTMs might enhance or inhibit antibody binding

How can sample preparation be optimized for different plant tissues?

Different plant tissues require adjusted extraction protocols for optimal CXE3 detection:

All buffers should be supplemented with a protease inhibitor cocktail immediately before use.

What are common causes of weak or absent signals in CXE3 detection?

Several factors may contribute to weak or absent signals when using CXE3 antibody:

• Insufficient protein concentration: Increase the amount of protein loaded or concentrate your sample.

• Protein degradation: Ensure complete protease inhibition during extraction and handle samples at 4°C.

• Inefficient transfer: Optimize transfer conditions based on protein size (~40 kDa for CXE3).

• Suboptimal antibody concentration: Titrate antibody concentrations to determine optimal working dilution.

• Epitope masking: Try alternative extraction buffers or denaturing conditions.

• Expired or degraded antibody: Use freshly thawed aliquots stored at recommended temperatures.

• Low expression levels: CXE3 expression may be condition-dependent; verify expression using RT-PCR.

When troubleshooting, change only one variable at a time and include positive controls (such as the provided recombinant immunogen) to validate your detection system .

How can background issues be minimized in immunodetection?

High background is a common challenge when using polyclonal antibodies like the CXE3 antibody. To reduce background:

• Increase blocking concentration (try 5-10% BSA or milk) and duration (2-3 hours)

• Add 0.1-0.3% Tween-20 to washing buffers and antibody dilution buffers

• Pre-absorb the antibody with proteins from negative control tissues

• Increase washing duration and number of washes (5-6 washes of 5-10 minutes each)

• Reduce primary and secondary antibody concentrations

• Include 5% normal serum (from the species of secondary antibody) in blocking buffer

• Filter all buffers before use to remove particulates

• For Western blots, consider using PVDF membranes instead of nitrocellulose

How can I validate antibody specificity for CXE3 detection?

Validating antibody specificity is essential for reliable research results. For CXE3 antibody:

• Perform peptide competition assays: Pre-incubate the antibody with excess recombinant CXE3 protein (provided in the kit) before application to your sample; signal disappearance confirms specificity.

• Test on multiple sample types: Compare CXE3 detection across tissues with known differential expression.

• Genetic validation: Test samples from CXE3 knockout or knockdown lines alongside wild-type controls.

• Compare with alternative detection methods: Correlate protein detection with mRNA levels via qRT-PCR.

• Immunoprecipitation followed by mass spectrometry: Confirm identity of proteins pulled down by the antibody.

• Use the pre-immune serum (provided in the kit) as a negative control to identify non-specific binding.

What considerations are important for quantitative analysis of CXE3?

For accurate quantitative analysis of CXE3 protein:

• Include a standard curve using recombinant CXE3 protein at known concentrations

• Ensure samples are within the linear range of detection

• Normalize CXE3 signals to appropriate loading controls (actin, tubulin, or total protein stain)

• Process all samples for comparison under identical conditions

• Perform at least three biological replicates and technical duplicates

• Use image analysis software that can account for background and perform accurate densitometry

• Apply appropriate statistical tests based on experimental design and data distribution

• For ELISA applications, prepare standard curves in the same matrix as experimental samples

How does CXE3 expression vary across plant development stages?

CXE3 expression patterns vary throughout Arabidopsis development, which has implications for experimental design. While comprehensive expression data specifically for CXE3 is limited in the search results, researchers should consider:

• Tissue-specific expression: Highest expression levels may be observed in metabolically active tissues

• Developmental regulation: Expression may change during seed germination, flowering, and senescence

• Stress responses: Exposure to xenobiotics, pathogens, or abiotic stresses may induce expression changes

When designing experiments, collect samples at consistent developmental stages and growth conditions to minimize variability. Time-course experiments capturing multiple developmental stages can provide valuable insights into CXE3 regulation.

How can I interpret contradictory results between different detection methods?

When faced with contradictory results between different detection methods (e.g., Western blot vs. ELISA):

• Consider protein conformation: Western blotting detects denatured proteins, while ELISA typically detects native conformations

• Epitope accessibility: Different sample preparations may expose or mask epitopes

• Sensitivity differences: ELISA is generally more sensitive than Western blotting

• Cross-reactivity profiles: Different techniques may have different cross-reactivity patterns

• Quantitative vs. qualitative results: Evaluate whether the contradictions are in protein presence/absence or in quantitative levels

• Technical variables: Buffer conditions, blocking agents, and detection systems differ between methods

To resolve contradictions, employ a third methodology (such as immunoprecipitation or immunohistochemistry) and correlate with gene expression data when possible.

What are appropriate experimental designs for studying CXE3 function?

When designing experiments to study CXE3 function:

• Genetic approaches:

  • Generate or obtain CXE3 knockout/knockdown lines

  • Create CXE3 overexpression lines

  • Use CRISPR/Cas9 for precise gene editing

• Biochemical approaches:

  • Enzyme activity assays with purified protein or plant extracts

  • Substrate specificity tests with various ester compounds

  • Inhibitor studies to characterize catalytic properties

• Expression analysis:

  • Spatial and temporal expression patterns using reporter constructs

  • Response to environmental stimuli and stressors

  • Co-expression analysis with related genes

• Protein interaction studies:

  • Co-immunoprecipitation using CXE3 antibody

  • Yeast two-hybrid or split-GFP assays for interaction partners

  • Subcellular localization studies

How do environmental conditions affect CXE3 expression and detection?

Environmental factors can significantly impact CXE3 expression and detection:

• Stress conditions: Exposure to cold, heat, drought, or high salinity may alter expression

• Chemical treatments: Herbicides, pesticides, or other xenobiotics may induce expression as part of detoxification responses

• Pathogen exposure: Biotic stresses may trigger defense responses involving CXE3

• Growth conditions: Light intensity, photoperiod, and nutrient availability can modulate expression

• Developmental timing: Sampling at different times of day or developmental stages may yield different results

For consistent results, maintain strictly controlled growth conditions across experiments and document all environmental parameters. When studying environmental responses, include time-course analyses to capture the dynamics of expression changes.

How can CXE3 antibody be used in comparative studies across plant species?

When using CXE3 antibody for cross-species studies:

• Sequence homology: Perform bioinformatic analysis to identify CXE3 homologs in target species

• Epitope conservation: Assess conservation of antibody recognition sites using sequence alignment

• Validation steps: Perform preliminary Western blots with positive controls from Arabidopsis alongside samples from target species

• Optimization: Adjust antibody concentrations, incubation times, and detection methods for each species

• Confirmation: Use alternative methods (gene expression, activity assays) to corroborate protein detection results

The antibody is raised against Arabidopsis thaliana CXE3 , so its reactivity may vary with evolutionary distance from this model organism.

What are the implications of CXE3 post-translational regulation for experimental design?

Post-translational regulation of CXE3 has important implications for research:

• Protein stability: CXE3 may have variable half-lives depending on cellular conditions

• Activity regulation: Phosphorylation or other modifications may alter enzymatic activity without changing protein levels

• Localization changes: Modifications may affect subcellular targeting

• Protein-protein interactions: PTMs may mediate dynamic interaction networks

To account for these factors:

• Compare protein levels with enzymatic activity measurements

• Consider subcellular fractionation in addition to whole-cell extracts

• Use phosphatase inhibitors in extraction buffers when studying phosphorylation

• Include time-course analyses to capture dynamic regulation

How can I integrate CXE3 protein data with transcriptomic and metabolomic analyses?

Integrating multiomics data for comprehensive CXE3 research:

• Correlation analysis: Compare CXE3 protein levels with mRNA expression patterns

• Pathway mapping: Overlay CXE3 expression with metabolite profiles of potential substrates and products

• Network analysis: Identify co-regulated genes and metabolites across conditions

• Temporal dynamics: Analyze time-course data to establish cause-effect relationships

• Statistical integration: Use multivariate statistical methods to identify patterns across data types

This integrated approach can provide insights into CXE3 function that might be missed by single-omics approaches, particularly for understanding its role in metabolic pathways.