EXL6 Antibody

What is the X6 antibody and what is its primary research application?

The X6 antibody is a novel anti-gliadin antibody specifically developed for gluten quantification in food samples. It recognizes the QXQPFPXP epitope sequence, which is critical in binding to cereal prolamins involved in celiac disease manifestation. The antibody demonstrates high specificity for wheat, barley, and rye proteins, making it particularly valuable for celiac disease research and food safety analysis . Its primary application is in ELISA-based detection and quantification of gluten content in various food matrices, providing researchers with a reliable tool for investigating gluten contamination and celiac-related protein interactions.

What is the ELOVL6 antibody and what biological systems does it target?

The ELOVL6 antibody is a rabbit polyclonal antibody specifically designed to target human ELOVL6 (Elongation of very long chain fatty acids protein 6). This antibody has been validated for immunohistochemistry (IHC) applications and has undergone enhanced validation procedures to ensure specificity and reproducibility . ELOVL6 is involved in fatty acid elongation, particularly the conversion of palmitic acid to stearic acid, making this antibody valuable for researchers investigating lipid metabolism, fatty acid synthesis pathways, and related metabolic disorders.

What validation methods have been applied to ensure EXL6 antibody specificity?

For the X6 antibody, specificity validation has been conducted through immunoblotting studies demonstrating selective recognition of wheat, barley, and rye proteins, as well as α-gliadin homologs from non-edible cereals like Dasypyrum villosum. Additionally, epitope mapping confirmed the QXQPFPXP recognition site as the primary binding target. ELISA correlation studies were performed comparing X6 performance with established antibodies like R5 and G12, showing high statistical correlation (Pearson's R = 0.86 and 0.87, respectively) .

For the ELOVL6 antibody, validation has been performed for IHC applications through a standardized process that ensures rigorous quality control. The enhanced validation approach likely includes Western blot analysis, peptide array testing, and immunohistochemistry on relevant tissues to confirm target specificity .

How does the X6 antibody compare methodologically to established antibodies in gluten detection?

The X6 antibody demonstrates comparable performance to established standards like R5 and G12 antibodies, which are recognized by Codex Alimentarius for gluten quantification. Statistical analysis shows high correlation between X6-based ELISA and both R5 and G12 methods (Pearson's R = 0.86 and 0.87, respectively), with no significant differences in qualitative assessment . Methodologically, X6 offers an advantage through its specific recognition of the QXQPFPXP epitope, which is present in proteins involved in celiac disease pathogenesis. Researchers should note that when implementing X6 in ELISA protocols, similar procedural conditions to those used with R5 and G12 can be applied, including substrate development time (15 min at room temperature) and washing procedures as detailed in the research protocol .

What is the molecular basis for X6 antibody specificity in cereal protein recognition?

The molecular basis for X6 antibody specificity lies in its recognition of the QXQPFPXP epitope sequence, with the PFP motif being most critical for binding. This explains the observed pattern of reactivity across different cereal proteins:

• Strong recognition of wheat, barley, and rye proteins, which contain the complete epitope including the critical PFP motif

• Reduced reactivity to avenin (oat protein), which lacks the PFP motif

• No recognition of proteins from Zea mays (corn) and Setaria italica (foxtail millet), which lack the required epitope structure

This molecular specificity allows researchers to use X6 antibody to selectively detect proteins implicated in celiac disease while excluding non-reactive cereals that are considered safe for celiac patients.

How can the X6 antibody be utilized in studies of celiac disease pathogenesis beyond basic gluten detection?

Beyond basic gluten detection, the X6 antibody offers valuable research applications for investigating celiac disease pathogenesis. Researchers can employ X6 in immunoprecipitation studies to isolate specific gliadin fragments containing the QXQPFPXP epitope from complex food matrices or biological samples. This approach allows for downstream mass spectrometry analysis to characterize peptide modifications that may affect immunogenicity. Additionally, X6 can be utilized in immunohistochemical analysis of intestinal biopsy samples to study the binding and localization of gluten peptides in tissue microenvironments . By coupling X6 antibody with immune cell response assays, researchers can investigate the relationship between epitope recognition and T-cell activation in celiac patients, potentially leading to new insights into disease mechanisms and therapeutic targets.

What considerations should researchers take into account when using ELOVL6 antibody for metabolic research?

When using ELOVL6 antibody for metabolic research, researchers should consider several important factors. First, the tissue-specific expression patterns of ELOVL6 vary significantly, with highest expression typically observed in liver, adipose tissue, and brain. Second, metabolic state (fasting, feeding, insulin stimulation) can dramatically alter ELOVL6 expression levels, requiring careful experimental design with appropriate controls and timing considerations. Third, species differences in ELOVL6 expression and regulation are significant; while the antibody is validated against human ELOVL6, researchers working with animal models should validate cross-reactivity and specificity in their specific model system . Finally, researchers should consider parallel assessments of related fatty acid metabolism enzymes (such as SCD1, FASN, and other ELOVL family members) to comprehensively understand the metabolic context of their findings.

What advanced experimental approaches can be used to map the complete binding characteristics of the X6 antibody?

Advanced experimental approaches for comprehensive mapping of X6 antibody binding characteristics include:

These approaches would provide researchers with comprehensive binding profiles to optimize experimental design and interpret results with greater confidence.

How should researchers address cross-reactivity concerns when using the X6 antibody in complex food matrices?

When working with complex food matrices, researchers should implement several strategies to address potential X6 antibody cross-reactivity issues:

• Include appropriate extraction controls using known gluten-free and gluten-containing samples processed alongside test samples

• Perform competitive inhibition ELISA with purified peptides containing the QXQPFPXP epitope to confirm specificity of detection

• Consider complementary testing with alternative antibodies (such as R5 or G12) that recognize different epitopes to corroborate findings

• For samples with high protein content from non-gluten sources, implement additional sample clean-up steps to reduce background signal

• When analyzing fermented food products, account for potential epitope degradation through parallel testing of hydrolyzed and non-hydrolyzed samples

For definitive analysis in particularly complex matrices, confirmation using mass spectrometry techniques targeting gluten-specific peptides can provide orthogonal validation of antibody-based findings.

What are the critical factors affecting reproducibility in ELOVL6 antibody-based experiments?

Several critical factors influence reproducibility in ELOVL6 antibody-based experiments:

• Tissue fixation and processing methods: Overfixation can mask epitopes, while inadequate fixation may compromise tissue morphology

• Antigen retrieval protocol: The specific pH, buffer composition, and temperature/pressure conditions significantly impact epitope accessibility

• Antibody concentration: The recommended concentration of 0.05 mg/ml should be optimized for specific applications and tissue types

• Detection system sensitivity: Signal amplification methods should be selected based on expected expression levels

• Sample storage conditions: Degradation of phosphorylation sites or protein modification can occur during improper storage

• Standardized positive and negative controls: These should be included in each experimental batch to normalize between runs

Researchers should maintain detailed records of these parameters to ensure consistent results across experiments and facilitate troubleshooting when variability is observed.

How can researchers optimize ELISA protocols specifically for the X6 antibody to improve sensitivity and specificity?

To optimize ELISA protocols specifically for the X6 antibody, researchers should consider the following methodological refinements:

• Coating buffer optimization: Test pH ranges from 9.3-9.8 for maximal antigen binding while maintaining epitope accessibility

• Blocking agent selection: Compare casein-based blockers (as used in validation studies) with alternatives like BSA or commercial blocking buffers to minimize background while preserving specific signal

• Sample extraction protocol: Optimize ethanol concentration (typically 60-70%) and extraction time to maximize recovery of target proteins

• Incubation conditions: Standard protocol uses 37°C for 30 minutes for primary antibody binding, but extension to 60 minutes at reduced temperature (25°C) may improve sensitivity for some sample types

• Washing stringency: Implement a 5-time washing procedure with detergent-containing buffer to reduce non-specific binding while preserving specific interactions

• Signal development optimization: The standard 15-minute development time at room temperature can be adjusted based on specific substrate systems and detection limits required

A systematic optimization approach testing these variables will allow researchers to develop a robust, sensitive ELISA protocol tailored to their specific research requirements.

How does the X6 antibody compare with R5 and G12 antibodies in detecting specific cereal proteins in research applications?

The X6 antibody exhibits distinct recognition patterns compared to R5 and G12 antibodies, which are important considerations for research applications:

While all three antibodies show high correlation in quantitative assessment (Pearson's R = 0.86-0.87 between X6 and R5/G12), qualitative assessment revealed no significant differences in detection capabilities across wheat, barley, and rye proteins . For research focusing specifically on peptides containing the PFP motif, X6 may offer advantages in epitope-specific studies, while R5 and G12 continue to serve as well-established reference methods with extensive validation data.

What methodological approaches can researchers use to integrate ELOVL6 antibody data with transcriptomics and metabolomics findings?

To effectively integrate ELOVL6 antibody-based protein expression data with transcriptomics and metabolomics findings, researchers can employ several methodological approaches:

• Parallel sample processing: Extract protein, RNA, and metabolites from the same biological samples to minimize variability

• Correlation analysis: Calculate Spearman or Pearson correlation coefficients between ELOVL6 protein expression levels (quantified from immunoblots or IHC), mRNA expression (from RNA-seq or qPCR), and relevant fatty acid metabolites (from LC-MS/MS)

• Pathway enrichment analysis: Integrate protein expression data with transcriptomics using tools like Ingenuity Pathway Analysis or GSEA to identify co-regulated networks

• Time-course experiments: Analyze temporal relationships between transcriptional activation, protein expression, and metabolite alterations following experimental interventions

• Single-cell/spatial approaches: Combine single-cell transcriptomics with spatial proteomics using the ELOVL6 antibody to understand cellular heterogeneity in metabolic responses

• Computational modeling: Develop mathematical models incorporating ELOVL6 enzyme kinetics with transcriptional regulation and metabolic flux data

This multi-omics approach provides a comprehensive understanding of ELOVL6 regulation and function in specific biological contexts.

How can researchers design experiments to address discrepancies between antibody-based detection and mass spectrometry results when studying gluten proteins?

When addressing discrepancies between X6 antibody-based detection and mass spectrometry results for gluten proteins, researchers should implement a systematic experimental design:

• Sample preparation comparison:

  • Process identical samples in parallel for both antibody-based and MS-based detection

  • Compare extraction efficiencies using different solvent systems (e.g., 60% ethanol vs. 50% propanol)

  • Evaluate the impact of reduction and alkylation steps on epitope accessibility

• Epitope-focused analysis:

  • Target the specific QXQPFPXP epitope region in MS analysis through custom database searches

  • Perform immunoprecipitation with X6 antibody followed by MS analysis of bound proteins

  • Consider potential post-translational modifications that might affect antibody recognition but be detectable by MS

• Quantification methodology:

  • Implement absolute quantification using isotopically labeled standards for both approaches

  • Develop calibration curves with purified gliadin standards analyzed by both methods

  • Calculate limits of detection and quantification for each approach to determine sensitivity differences

• Data integration strategy:

  • Apply normalization procedures to allow direct comparison between methods

  • Develop computational approaches to reconcile datasets, accounting for known biases in each method

  • Implement machine learning algorithms to predict antibody recognition based on MS-derived sequence information

This comprehensive approach not only resolves discrepancies but also leverages the complementary strengths of both techniques to generate more robust and reliable data.