LTC4S Antibody

What is the molecular function of LTC4S and why is it important in research?

LTC4S (Leukotriene C4 Synthase) is an enzyme that catalyzes the conjugation of leukotriene A4 with reduced glutathione (GSH) to form leukotriene C4 with high specificity. Additionally, it can catalyze the transfer of a glutathionyl group from GSH to 13(S),14(S)-epoxy-docosahexaenoic acid to form maresin conjugate in tissue regeneration 1 (MCTR1), which possesses potent anti-inflammatory and proresolving actions . The enzyme is approximately 16.6 kDa in molecular weight and is primarily localized in the nucleus outer membrane, endoplasmic reticulum membrane, and functions as a multi-pass membrane protein .

Research interest in LTC4S has increased significantly with recent findings showing its potential role in lung adenocarcinoma, where its expression levels correlate with patient prognosis. Understanding LTC4S regulation and function can provide insights into inflammatory pathways and potentially novel therapeutic targets for cancer treatment .

What types of LTC4S antibodies are available for research applications?

Currently, researchers have access to several types of LTC4S antibodies optimized for different experimental applications:

Both commercially available antibodies are unconjugated IgG isotype and have been affinity-purified for research use . The choice between these antibodies depends on the specific research requirements, including species of interest and desired application.

What are the optimal storage conditions and handling procedures for LTC4S antibodies?

For maximum stability and activity retention, LTC4S antibodies should be stored at -20°C for up to 1 year from the date of receipt . The antibodies are typically supplied in a liquid formulation containing PBS with additives such as 50% glycerol, 0.5% BSA, and 0.02% sodium azide .

To maintain antibody integrity:

• Avoid repeated freeze-thaw cycles which can degrade antibody performance

• Store in small aliquots if frequent use is anticipated

• Some suppliers recommend storing sealed kits at 2-8°C, with specific components requiring different storage conditions after opening

• Allow antibodies to equilibrate to room temperature before opening for use

Following these storage recommendations ensures maximum shelf-life and consistent experimental results in detection applications.

What dilution ranges are recommended for different LTC4S antibody applications?

The optimal dilution of LTC4S antibodies varies by application and specific antibody formulation:

Researchers should note that these are starting recommendations, and optimal dilutions should be determined experimentally for each specific research condition. Factors affecting optimal dilution include sample type, protein expression levels, and detection method sensitivity .

How does post-translational modification of LTC4S affect antibody detection and enzyme function?

Post-translational modifications significantly impact LTC4S function and should be considered when selecting antibodies and interpreting results. Notably, phosphorylation at Ser-36 by RPS6KB1 (p70S6K) has been demonstrated to inhibit the leukotriene-C4 synthase activity . This site-specific modification occurs within the 29-79 amino acid region targeted by some commercially available antibodies.

When designing experiments:

• Consider whether phosphorylation state might affect antibody binding affinity

• For studies investigating LTC4S regulation, phospho-specific antibodies may be required

• Treatment with phosphatases before immunoblotting might provide insights into total protein levels versus phosphorylated forms

• Correlation between phosphorylation status and enzymatic activity should be experimentally validated

Researchers investigating signaling pathways affecting LTC4S should particularly note this regulatory mechanism and consider how experimental conditions might alter phosphorylation status, potentially leading to variation in antibody detection efficiency.

What methodological approaches can be used to study LTC4S expression in lung adenocarcinoma research?

Recent studies have identified LTC4S as potentially significant in lung adenocarcinoma (LUAD), with its expression levels correlating with patient prognosis . When investigating LTC4S in LUAD research, several methodological approaches are recommended:

• Gene Expression Analysis: Public datasets like TCGA-LUAD and GEO (GSE50081, GSE30219) can be utilized to examine LTC4S expression. In GSE50081, a median expression value of 162 was used as the cutoff to define high versus low expression groups, while in GSE30219, the cutoff was 71 .

• Methylation Analysis: DNA methylation profiling using platforms like Illumina Infinium HumanMethylation450 can reveal epigenetic regulation of LTC4S. TCGA lung adenocarcinoma methylation data suggests hypermethylation may be involved in LTC4S downregulation, potentially mediated by DNMT3A .

• Protein Detection: Western blotting with specific anti-LTC4S antibodies can validate expression findings at the protein level. Recommended dilution ranges of 1:500-2000 should be optimized for lung tissue or cell line lysates .

• Functional Studies: For mechanistic investigations, gain-of-function assays in LUAD cell lines (A549, NCI-H1299) followed by xenograft models can establish the functional significance of LTC4S .

• Pathway Analysis: Gene Set Enrichment Analysis (GSEA) can identify LTC4S-associated pathways, including HALLMARK and KEGG signaling pathways, providing insights into the molecular mechanisms of LTC4S in LUAD progression .

What are the technical considerations for using LTC4S antibodies in sandwich ELISA applications?

When implementing sandwich ELISA for LTC4S detection, researchers should consider several technical factors to ensure reliable and reproducible results:

The commercially available Human LTC4S ELISA Kit employs a double antibody-sandwich method with the following technical specifications:

For optimal results:

• The microplate is pre-coated with anti-LTC4S antibody

• Add standards and properly diluted samples to the appropriate wells

• After incubation, wash to remove unbound components

• Add biotinylated detection antibody which binds to LTC4S

• Add HRP-Streptavidin Conjugate (SABC) after washing

• Add TMB substrate solution which is catalyzed by HRP to produce a blue color

• Add stop solution to turn the reaction yellow

• Measure absorbance at 450 nm and calculate concentration using the standard curve

The concentration of LTC4S in samples is directly proportional to the OD450 value, allowing precise quantification within the assay's detection range.

How can researchers troubleshoot specificity issues with LTC4S antibodies?

Ensuring antibody specificity is crucial for generating reliable research data. For LTC4S antibodies, consider the following troubleshooting approaches:

• Validation Controls:

  • Include positive controls from tissues known to express LTC4S (e.g., lung or immune cells)

  • Use recombinant LTC4S protein as a positive control

  • Include samples from LTC4S knockout models as negative controls where available

• Cross-Reactivity Assessment:

  • Review the antibody's cross-reactivity profile with related proteins

  • The anti-LTC4S antibody (29-79 aa) detects endogenous levels of LTC4S in human, mouse, and rat samples

  • Specific ELISA kits claim to bind specifically with LTC4S with no obvious cross-reaction with other analogues

• Blocking Peptide Competition:

  • Pre-incubate the antibody with the immunizing peptide (e.g., synthetic peptide from amino acids 29-79 or 29-55)

  • Compare detection patterns between blocked and unblocked antibody

  • Specific binding should be competitively inhibited by the immunizing peptide

• Multiple Detection Methods:

  • Confirm findings using antibodies targeting different epitopes

  • Correlate protein detection with mRNA levels using RT-PCR

  • Consider mass spectrometry validation for absolute confirmation

• Antibody Validation:

  • Review if the antibody was affinity-purified using epitope-specific immunogen

  • Check if the supplier performed peptide affinity purification

  • Assess if the antibody has been validated in publications for your specific application

What are the potential roles of LTC4S in cancer biology beyond lung adenocarcinoma?

While recent research has highlighted LTC4S downregulation in lung adenocarcinoma , its broader roles in cancer biology merit investigation:

LTC4S catalyzes the production of leukotriene C4, a key inflammatory mediator in the arachidonic acid pathway. This pathway has multiple connections to cancer biology:

• Inflammation and Cancer Progression: Leukotrienes generally promote pro-inflammatory environments that can contribute to tumor promotion and progression. LTC4S expression changes may alter the inflammatory milieu surrounding tumors.

• Resolution of Inflammation: LTC4S can also catalyze the formation of maresin conjugate in tissue regeneration 1 (MCTR1), which possesses anti-inflammatory and pro-resolving properties . Dysregulation of these resolution pathways might contribute to chronic inflammation and cancer development.

• Epigenetic Regulation: The hyper-methylation and DNMT3A-mediated downregulation of LTC4S observed in lung adenocarcinoma suggests epigenetic mechanisms may control its expression in other cancer types as well.

• mTORC1 Signaling Interaction: Research indicates potential crosstalk between LTC4S and mTORC1 signaling in lung adenocarcinoma , a pathway frequently dysregulated in multiple cancer types.

• Tumor Microenvironment Modulation: As LTC4S products influence inflammatory cell recruitment and function, its expression may shape the tumor immune microenvironment.

Researchers investigating LTC4S in cancer contexts should consider these multiple potential roles and design experiments to distinguish between them, potentially using LTC4S antibodies to quantify expression across different cancer types and correlate with clinical outcomes.