CXE15 Antibody

What is CXE15 and why are antibodies against it valuable in research?

CXE15 (carboxylesterase 15) is an enzyme in Arabidopsis thaliana that plays a crucial role in depleting strigolactones, which are phytohormones that coordinate various growth and developmental processes . Antibodies against CXE15 are valuable research tools for:

• Detecting protein expression levels in different plant tissues

• Immunoprecipitation experiments to study protein-protein interactions

• Immunohistochemistry to determine spatial localization

• Western blotting to quantify expression under various conditions

• Tracking post-translational modifications

The N-terminal region of CXE15 is characterized by an alpha helix structure, which differs from related enzymes like CXE20 (which displays an alpha/beta fold) . This structural distinction makes specific antibody detection particularly valuable for distinguishing between these related carboxylesterases.

What sample types are suitable for CXE15 antibody detection?

CXE15 antibodies can be applied to various sample types, similar to other plant protein antibodies:

When preparing samples, it's critical to consider the subcellular localization of CXE15 and use appropriate extraction methods to ensure efficient protein recovery while maintaining the epitope integrity for antibody recognition.

What controls should be included when using CXE15 antibodies?

Proper controls are essential for interpreting antibody-based experimental results:

• Positive Control: Samples known to express CXE15 (e.g., tissues where strigolactone catabolism is active)

• Negative Control: Samples from CXE15 knockout plants or tissues known not to express CXE15

• Isotype Control: Non-specific antibody of the same isotype to assess background binding

• Blocking Peptide Control: Pre-incubation of antibody with the immunizing peptide to confirm specificity

• Secondary Antibody Control: Omitting primary antibody to assess non-specific binding of secondary antibody

Similar to validation approaches used with other antibodies, researchers should consider using genetic knockouts of CXE15 as the gold standard negative control, as demonstrated with CXCR5 antibody validation where knockout cell lines were employed .

How can CXE15 antibodies be used to study the relationship between strigolactone signaling and plant development?

CXE15 antibodies enable sophisticated investigations into strigolactone (SL) regulation:

• Developmental Expression Profiling: Using immunoblotting to track CXE15 expression across developmental stages to correlate with SL-dependent phenotypes

• Environmental Response Studies: Quantifying CXE15 expression changes under different environmental conditions (drought, nutrient availability, light conditions) to understand SL catabolism regulation

• Co-localization Studies: Combining CXE15 antibodies with markers for subcellular compartments to determine where SL degradation occurs

• Protein Complex Identification: Using co-immunoprecipitation with CXE15 antibodies to identify interaction partners involved in SL metabolism

Research shows that understanding CXE15 expression patterns can provide insights into when and where plants actively regulate SL levels, as CXE15 participates in post-signaling processes for signal inactivation and cue depletion .

What methods can be used to assess CXE15 enzyme activity in relation to antibody detection?

Correlating antibody detection with enzyme activity provides more comprehensive insights:

When designing such experiments, it's important to consider that antibody binding might affect enzyme activity, particularly if the epitope is near the catalytic site. The crystal structures of AtCXE15, both in apo form and with bound SL intermediate , can guide researchers in selecting antibodies that target accessible epitopes without interfering with catalytic function.

How can phosphorylation states of CXE15 be studied using specialized antibodies?

While standard CXE15 antibodies detect the protein regardless of phosphorylation status, phospho-specific antibodies can provide insights into regulatory mechanisms:

• Phospho-specific Antibody Development: Generate antibodies against predicted phosphorylation sites in CXE15 based on consensus sequences and structural accessibility

• 2D Gel Electrophoresis + Immunoblotting: Separate proteins by charge and size to detect phosphorylated variants using standard CXE15 antibodies

• Phos-tag™ SDS-PAGE: Utilize phosphate-binding molecules to create mobility shifts in phosphorylated proteins, detectable with standard CXE15 antibodies

• Combined Phospho-enrichment and Antibody Detection: Use titanium dioxide or IMAC to enrich phosphopeptides prior to detection

Understanding CXE15 phosphorylation may reveal regulatory mechanisms controlling its catalytic activity on strigolactones, providing insights into how plants fine-tune phytohormone levels during development and in response to environmental cues.

What are the optimal conditions for immunoprecipitation experiments using CXE15 antibodies?

Successful immunoprecipitation of CXE15 requires careful optimization:

Given that CXE15 has distinct structural features including an alpha helix in its N-terminal region , researchers should consider whether the antibody epitope remains accessible when the protein is in complex with interaction partners.

How should researchers optimize western blotting protocols for CXE15 detection?

Optimizing western blotting for CXE15 requires attention to several parameters:

• Sample Preparation: Consider using specialized plant protein extraction buffers containing reducing agents to maintain protein integrity

• Gel Percentage: 10-12% polyacrylamide gels typically provide optimal resolution for CXE15 (~42-45 kDa)

• Transfer Conditions: Semi-dry transfer at 15-20V for 30-45 minutes or wet transfer at 30V overnight at 4°C

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

• Antibody Dilution: Start with 1:1000 dilution and optimize based on signal-to-noise ratio

• Detection Method: Enhanced chemiluminescence for standard detection or fluorescent secondary antibodies for quantitative analysis

Similar to approaches used with CXCR5 antibodies , performing western blots under reducing conditions with appropriate controls is essential for ensuring specificity. Researchers should also note that boiling samples may cause protein aggregation in some cases, as observed with certain membrane-associated proteins .

What considerations are important when using CXE15 antibodies for immunohistochemistry in plant tissues?

Plant tissues present unique challenges for immunohistochemistry:

• Fixation: 4% paraformaldehyde is generally suitable; avoid glutaraldehyde which can create high autofluorescence

• Permeabilization: Use 0.1-0.3% Triton X-100 to facilitate antibody penetration through cell walls

• Antigen Retrieval: Heat-mediated retrieval with citrate buffer (pH 6.0) can improve epitope accessibility

• Background Reduction: Pre-incubate sections with 10% normal serum from the species in which the secondary antibody was raised

• Signal Amplification: Consider tyramide signal amplification for low-abundance targets

• Counterstaining: Use DAPI for nuclei and specific organelle markers to provide context for CXE15 localization

Drawing from techniques used for other antibodies , incubation time optimization (10 minutes to overnight) can significantly impact staining quality and specificity.

How can researchers troubleshoot non-specific binding or weak signals with CXE15 antibodies?

When encountering detection problems with CXE15 antibodies:

Learning from antibody validation approaches in other systems , generating and including a CXE15 knockout sample as a negative control provides the strongest evidence for antibody specificity.

What are the best approaches for quantifying CXE15 expression levels across different experimental conditions?

Accurate quantification requires rigorous methodology:

• Normalization Strategy:

  • Use multiple housekeeping proteins as internal controls

  • Consider tissue-specific reference genes for RT-qPCR validation

  • Include loading controls on every blot

• Image Acquisition:

  • Use dynamic range-appropriate exposure settings

  • Avoid saturated pixels that compress signal differences

  • Capture multiple exposures to ensure linearity

• Quantification Software:

  • Employ background subtraction with defined parameters

  • Use region-of-interest analysis with consistent sizing

  • Apply lane normalization based on total protein or housekeeping proteins

• Statistical Analysis:

  • Perform technical and biological replicates (minimum n=3)

  • Apply appropriate statistical tests based on data distribution

  • Report both fold change and p-values

Following practices like those used in validating CXCR5 antibodies , collecting >5000 events for flow cytometry or using multiple technical repeats for western blotting helps ensure robust quantitative data.

How can researchers distinguish between CXE15 and related carboxylesterases (such as CXE20) when using antibodies?

Distinguishing between related carboxylesterases requires strategic approaches:

• Epitope Selection: Choose antibodies targeting unique regions, particularly focusing on the N-terminal differences between CXE15 (alpha helix) and CXE20 (alpha/beta fold)

• Validation Testing: Test antibodies against recombinant CXE15 and CXE20 to confirm specificity

• Pre-absorption Controls: Pre-incubate antibody with recombinant CXE20 to remove cross-reactive antibodies before detecting CXE15

• Knockout Validations: Utilize genetic knockouts of either CXE15 or CXE20 to confirm antibody specificity

• Size Discrimination: While related, CXE15 and CXE20 may have slightly different molecular weights detectable on high-resolution gels

Similar to validation strategies used for other antibodies , analyzing the antibody's performance in both wild-type and knockout backgrounds provides definitive evidence of specificity.

How can CXE15 antibodies be used to study evolutionary conservation across plant species?

Cross-species applicability of CXE15 antibodies depends on epitope conservation:

• Sequence Alignment Analysis: Compare CXE15 sequences across species to identify conserved epitopes

• Cross-Reactivity Testing: Validate antibody binding in related species through western blotting

• Heterologous Expression Testing: Express CXE15 orthologs from different species and test antibody recognition

• Phylogenetic Correlation: Map antibody reactivity to evolutionary relationships between plant species

Antibodies targeting highly conserved catalytic domains may provide broader cross-species reactivity compared to those targeting variable regions, enabling comparative studies of CXE15 function across different plant families.

What methods are being developed to multiplex CXE15 detection with other strigolactone pathway components?

Advanced multiplexing approaches include:

Multiplexing allows researchers to study how CXE15 fits into the broader strigolactone regulatory network, providing insights into protein interaction dynamics and pathway regulation mechanisms that would be difficult to obtain through single-protein studies.