LTA4H Antibody, HRP conjugated

What is LTA4H and why is it important in biomedical research?

Leukotriene A4 Hydrolase (LTA4H) is a bifunctional enzyme with both epoxide hydrolase and aminopeptidase activities. The mature chain of human LTA4H consists of 610 amino acids (residues 2-611) and is highly specific for LTA4, which can also covalently modify and inhibit the enzyme . LTA4H has emerged as a significant research target due to its involvement in inflammatory processes and, more recently, its association with cancer development.

The importance of LTA4H in research has increased substantially following discoveries that it functions not only as a metabolic enzyme but also as an RNA-binding protein (RBP) involved in post-transcriptional control of specific mRNAs . This dual functionality positions LTA4H at the intersection of inflammatory pathways and gene regulation, making it a valuable target for both basic research and therapeutic development.

How does an HRP-conjugated LTA4H antibody differ from non-conjugated versions in research applications?

HRP-conjugated LTA4H antibodies contain horseradish peroxidase directly linked to the antibody molecule, eliminating the need for secondary antibody incubation steps in detection protocols. This conjugation provides several methodological advantages over non-conjugated antibodies:

• Direct detection capability that reduces protocol time and complexity

• Decreased background signal due to elimination of secondary antibody cross-reactivity

• Enhanced sensitivity for low-abundance targets through enzymatic amplification

• Compatibility with one-step detection systems in western blotting and immunohistochemistry

In practice, researchers typically use HRP-conjugated antibodies in western blot protocols by incubating membranes with the conjugated antibody for approximately 1 hour at room temperature, followed by direct visualization using chemiluminescent substrates . This streamlined approach contrasts with non-conjugated antibodies, which require a two-step incubation process with primary and secondary antibodies.

What are the optimal protocols for using LTA4H antibody, HRP conjugated in western blot applications?

For optimal western blot results using HRP-conjugated LTA4H antibodies, researchers should implement the following protocol:

• Sample Preparation:

  • Extract proteins from tissues or cells using RIPA buffer supplemented with protease inhibitors

  • Quantify proteins using Bradford or BCA assay to ensure equal loading

• Gel Electrophoresis and Transfer:

  • Separate 20-40 μg of protein on 10-12% SDS-PAGE gels

  • Transfer to PVDF or nitrocellulose membranes at 100V for 60-90 minutes

• Blocking and Antibody Incubation:

  • Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

  • Incubate with HRP-conjugated LTA4H antibody (1:1000 to 1:5000 dilution) for 1 hour at room temperature or overnight at 4°C

  • Wash thoroughly with TBST (3-5 washes, 5 minutes each)

• Detection:

  • Apply ECL substrate directly to membrane

  • Image using chemiluminescence detection system

  • Quantify band intensity using appropriate software (ImageJ, etc.)

Critical troubleshooting considerations include optimizing antibody concentration, ensuring adequate blocking to minimize background, and maintaining consistent washing procedures to preserve signal-to-noise ratio.

How can researchers validate the specificity of LTA4H antibody in experimental systems?

Validating antibody specificity is crucial for reliable research outcomes. For LTA4H antibodies, implement the following validation approaches:

• Positive and Negative Controls:

  • Use recombinant LTA4H protein as positive control

  • Include samples from LTA4H knockout models as negative controls

  • Compare expression in tissues known to have differential LTA4H expression

• Multiple Detection Methods:

  • Cross-validate western blot findings with immunohistochemistry

  • Perform immunoprecipitation followed by mass spectrometry

  • Compare results with orthogonal detection methods like qRT-PCR for mRNA expression

• Peptide Competition Assays:

  • Pre-incubate antibody with purified LTA4H peptide

  • Observe elimination of specific signal in subsequent detection

  • Quantify signal reduction to assess specificity

• siRNA Knockdown Verification:

  • Perform western blot on samples with LTA4H knockdown

  • Confirm reduction in signal correlates with knockdown efficiency

  • Validate with multiple siRNA constructs to rule out off-target effects

These validation steps ensure experimental results accurately reflect LTA4H biology rather than artifacts from non-specific antibody binding.

How is LTA4H antibody used to investigate its role in cancer progression and metastasis?

LTA4H antibodies have become instrumental in elucidating the enzyme's role in cancer pathogenesis through several advanced applications:

• Tumor Tissue Microarray Analysis:

  • LTA4H antibodies are applied to tumor tissue microarrays to correlate expression with clinical outcomes

  • Studies have demonstrated that high LTA4H expression correlates with poor prognosis in head and neck squamous cell carcinoma (HNSCC)

  • Immunohistochemical staining patterns reveal subcellular localization changes in malignant versus normal tissues

• Signaling Pathway Dissection:

  • Western blotting with LTA4H antibodies helps identify downstream effectors in cancer pathways

  • Research has shown LTA4H's involvement in proliferation pathways through its interaction with critical oncogenes

  • Co-immunoprecipitation using LTA4H antibodies reveals protein-protein interactions in cancer signaling networks

• In vivo Tumor Models:

  • LTA4H antibodies track enzyme expression in patient-derived xenograft (PDX) models

  • Immunohistochemical analysis of Apc^Min/+^ mouse models using LTA4H antibodies has demonstrated the enzyme's role in colorectal tumorigenesis

  • Correlation between LTA4H expression and Ki-67 staining provides insights into proliferation mechanisms

• Treatment Response Monitoring:

  • LTA4H antibodies are used to assess target engagement following LTA4H inhibitor treatment

  • Studies with bestatin have used LTA4H antibodies to confirm decreased pathway activation and reduced proliferation markers

  • Quantitative image analysis of immunostained samples provides biomarker data for treatment efficacy

These applications have collectively established LTA4H as a potential therapeutic target across multiple cancer types, particularly in colorectal and laryngeal cancers.

What methodological approaches address contradictory findings regarding LTA4H expression in different cancer types?

Contradictory findings regarding LTA4H in cancer research present significant challenges. Researchers should implement the following methodological approaches to address discrepancies:

• Standardized Quantification Methods:

  • Employ digital pathology with validated scoring algorithms

  • Use consistent thresholds for categorizing "high" versus "low" expression

  • Report quantitative metrics rather than subjective assessments

• Context-Specific Analysis:

  • Stratify samples by molecular subtypes within each cancer type

  • Analyze expression in relation to tumor microenvironment characteristics

  • Consider temporal dynamics of expression during disease progression

• Multi-omics Integration:

  • Correlate protein expression data with transcriptomic profiles

  • Analyze epigenetic modifications that might explain expression differences

  • Implement proteogenomic approaches to relate genetic alterations to protein function

• Functional Validation Studies:

  • Perform gain and loss of function experiments in appropriate model systems

  • Evaluate enzymatic activity in addition to expression levels

  • Test the effects of selective inhibitors across diverse cancer models

By implementing these approaches, researchers can reconcile seemingly contradictory findings and develop a more nuanced understanding of LTA4H's context-dependent roles in cancer biology.

How can researchers investigate LTA4H's RNA-binding properties using antibody-based techniques?

Recent discoveries have revealed LTA4H's unexpected function as an RNA-binding protein, requiring specialized methodological approaches:

• Improved RNA Immunoprecipitation (iRIP-seq):

  • Use LTA4H antibodies to immunoprecipitate protein-RNA complexes

  • Sequence captured RNAs to identify binding targets

  • This approach has revealed LTA4H extensively binds to mRNAs/pre-mRNAs and lncRNAs in cancer cells

• Protocol Optimization:

  • Cross-link cells with formaldehyde to preserve RNA-protein interactions

  • Lyse cells in specialized buffers containing RNase inhibitors

  • Use LTA4H antibodies (either native or epitope-tagged versions) for immunoprecipitation

  • Wash stringently to remove non-specific interactions

  • Reverse cross-links and isolate RNA for downstream analysis

• Control Strategies:

  • Include IgG control immunoprecipitations

  • Perform RNA-binding assays in LTA4H-depleted cells

  • Use CRISPR-edited cells expressing RNA-binding-deficient LTA4H mutants

• Data Analysis Approaches:

  • Apply ABLIRC method to identify LTA4H-bound peaks precisely

  • Look for enriched sequence motifs (such as the AAGG motif found in LTA4H binding peaks)

  • Perform GO analysis on bound transcripts to identify biological pathways (studies have shown enrichment in mitotic cell cycle, DNA repair, and RNA splicing-related pathways)

This methodology has led to the significant finding that LTA4H binds to cancer-associated genes including LTBP3, ROR2, EGFR, HSP90B1, and lncRNAs like NEAT1, providing new mechanistic insights into its role in carcinogenesis .

What technical challenges arise when using HRP-conjugated antibodies for studying RNA-protein interactions?

When using HRP-conjugated LTA4H antibodies to study RNA-protein interactions, researchers face several technical challenges that require specific solutions:

• Signal Interference Issues:

  • Challenge: HRP conjugation can interfere with antibody binding to RNA-protein complexes

  • Solution: Perform parallel experiments with conjugated and non-conjugated antibodies to ensure comparable RNA enrichment

  • Validation: Quantify RNA recovery using RT-qPCR for known targets to confirm antibody performance

• Cross-linking Complications:

  • Challenge: Formaldehyde cross-linking may mask epitopes recognized by the antibody

  • Solution: Optimize cross-linking conditions (time, concentration) specifically for LTA4H

  • Alternative: Consider UV cross-linking for direct RNA-protein interactions

• RNase Contamination Risks:

  • Challenge: HRP preparation may contain trace RNase activity

  • Solution: Include additional RNase inhibitors in all buffers

  • Quality Control: Verify RNA integrity after immunoprecipitation using bioanalyzer

• Detection Method Compatibility:

  • Challenge: Traditional RNA visualization methods may be affected by HRP activity

  • Solution: Include appropriate quenching steps before RNA isolation

  • Alternative Approach: Consider using epitope-tagged LTA4H with anti-tag antibodies for RNA studies

These technical considerations are essential for generating reliable data when investigating the emerging RNA-binding functions of LTA4H in cancer and inflammatory conditions.

How can LTA4H antibodies be utilized to evaluate the efficacy of LTA4H inhibitors in clinical samples?

LTA4H antibodies serve as critical tools for evaluating inhibitor efficacy in clinical research:

• Target Engagement Assessment:

  • Use western blotting with LTA4H antibodies to quantify protein levels before and after inhibitor treatment

  • Perform immunohistochemistry on paired pre- and post-treatment tissue samples

  • Correlate changes in LTA4H pathway activation with clinical response

• Pharmacodynamic Biomarker Development:

  • Measure LTB4 concentration in patient blood samples as a surrogate marker for LTA4H activity

  • Compare LTB4 levels with immunohistochemical LTA4H staining to establish correlation

  • Track temporal changes in biomarker levels during treatment cycles

• Patient Stratification Strategies:

  • Categorize patients based on baseline LTA4H expression using immunohistochemistry

  • Correlate expression levels with treatment response to identify responsive subgroups

  • Develop predictive algorithms integrating LTA4H status with other clinical parameters

• Resistance Mechanism Investigation:

  • Use LTA4H antibodies to assess protein expression in treatment-resistant samples

  • Identify alterations in LTA4H localization or post-translational modifications

  • Combine with RNA analysis to detect splice variants that might confer resistance

Research with bestatin (an LTA4H inhibitor) in colorectal cancer has demonstrated the utility of this approach, where treatment efficacy was evaluated through LTB4 concentration measurements and immunohistochemical analysis of the LTA4H pathway activation .

What are the methodological considerations for using LTA4H and its product LTB4 as prognostic biomarkers in cancer?

The development of LTA4H and LTB4 as prognostic biomarkers requires rigorous methodological approaches:

• Sample Collection Standardization:

  • Establish protocols for tissue preservation that maintain LTA4H integrity

  • Standardize blood collection timing relative to treatment cycles

  • Implement quality control metrics for sample processing

• Assay Development and Validation:

  • Tissue Analysis:

    • Optimize immunohistochemistry protocols with multiple LTA4H antibodies

    • Develop automated scoring systems to reduce inter-observer variability

    • Validate scoring against patient outcomes in retrospective cohorts

  • Blood Biomarker Analysis:

    • Validate ELISA or mass spectrometry methods for LTB4 quantification

    • Establish reference ranges in healthy and disease populations

    • Determine stability parameters for sample storage and processing

• Multivariate Analysis Frameworks:

  • Integrate LTA4H/LTB4 biomarkers with established prognostic factors

  • Develop statistical models that account for confounding variables

  • Validate in independent patient cohorts across different cancer types

• Clinical Implementation Strategies:

  • Determine optimal sampling frequency for longitudinal monitoring

  • Establish clinically relevant thresholds for intervention

  • Develop standard operating procedures for clinical laboratories

Research has demonstrated that LTA4H expression levels correlate with survival probability in colorectal cancer and head and neck squamous cell carcinoma, with high expression associated with poor prognosis . Similarly, LTB4 concentration in blood samples has shown promise as a biomarker for evaluating treatment efficacy and disease progression .

What are the most common technical issues when using LTA4H antibody, HRP conjugated, and how can they be resolved?

Researchers frequently encounter these technical challenges with HRP-conjugated LTA4H antibodies:

• High Background Signal:

  • Cause: Insufficient blocking or non-specific binding

  • Solution: Increase blocking time and concentration (5-10% blocking agent)

  • Alternative: Use different blocking agents (BSA, casein, or commercial alternatives)

  • Additional Step: Include 0.1-0.3% Tween-20 in wash buffers to reduce hydrophobic interactions

• Weak or Absent Signal:

  • Cause: Epitope masking or protein degradation

  • Solution: Optimize protein extraction method to preserve LTA4H structure

  • Alternative: Try different lysis buffers that maintain enzymatic activity

  • Verification: Include positive control samples with known LTA4H expression

• Multiple Bands or Unexpected Band Sizes:

  • Cause: Cross-reactivity, post-translational modifications, or splice variants

  • Solution: Validate with recombinant LTA4H and knockout controls

  • Analysis: Compare band patterns across multiple tissue types

  • Confirmation: Perform mass spectrometry to identify unexpected bands

• Signal Variability Between Experiments:

  • Cause: Antibody degradation or sample preparation inconsistency

  • Solution: Aliquot antibody to avoid freeze-thaw cycles

  • Quality Control: Include internal reference samples in each experiment

  • Standardization: Develop standard curves with recombinant protein

Implementation of these troubleshooting approaches ensures reliable and reproducible results when utilizing HRP-conjugated LTA4H antibodies in research applications.

How should researchers validate batch-to-batch consistency of LTA4H antibodies for longitudinal studies?

Ensuring antibody consistency is critical for longitudinal studies spanning months or years:

• Initial Characterization Protocol:

  • Perform titration experiments to determine optimal working concentration

  • Create standard curves using recombinant LTA4H protein

  • Document specific detection limits and linear range

  • Store reference images and quantification data as baseline

• Regular Quality Control Testing:

  • Maintain reference sample set (positive, negative, and gradient controls)

  • Test each new antibody batch against reference samples

  • Compare signal-to-noise ratio, EC50 values, and detection limits

  • Document lot-specific performance characteristics

• Cross-Validation Methods:

  • Use multiple detection methods (western blot, ELISA, IHC)

  • Perform parallel testing with previously validated antibody lots

  • Calculate correlation coefficients between old and new batches

  • Establish acceptance criteria based on statistical variability

• Long-term Reference Standards:

  • Create and preserve stable reference materials (lysates, tissue blocks)

  • Implement normalization procedures using housekeeping proteins

  • Maintain a dedicated antibody performance database

  • Develop mathematical correction factors for inter-batch variability

This systematic approach to antibody validation is essential for generating comparable data across extended research timelines, particularly in longitudinal clinical studies evaluating LTA4H as a biomarker or therapeutic target.

How can LTA4H antibodies be used to investigate the dual enzymatic and RNA-binding functions in different cellular contexts?

Recent discoveries about LTA4H's dual functionality open new research avenues requiring sophisticated methodological approaches:

• Domain-Specific Functional Analysis:

  • Use domain-specific LTA4H antibodies to distinguish between enzymatic and RNA-binding regions

  • Perform structure-function studies with mutant LTA4H constructs

  • Correlate RNA binding with enzymatic activity in various cellular contexts

• Cellular Compartmentalization Studies:

  • Employ subcellular fractionation followed by western blotting

  • Perform immunofluorescence with LTA4H antibodies to track localization changes

  • Correlate subcellular distribution with specific functions in different cell types

• Integrated Multi-omics Approach:

  • Combine RNA-seq, iRIP-seq, and proteomics data

  • Develop computational models of LTA4H's dual functionality

  • Map interaction networks specific to each function

• Translational Regulation Investigation:

  • Analyze polysome profiles with LTA4H antibodies

  • Perform RNA stability assays in the presence/absence of LTA4H

  • Investigate the role of LTA4H in stress granule formation during cellular stress

This integrated approach will help elucidate how LTA4H coordinates its enzymatic and RNA-binding functions, particularly in contexts where it influences cancer-related pathways like mitotic cell cycle, DNA repair, and RNA splicing .

What novel methodological approaches are emerging for studying LTA4H in the context of cancer immunotherapy?

The intersection of LTA4H biology with cancer immunology presents exciting research opportunities:

• Immune Cell Infiltration Analysis:

  • Multiplex immunohistochemistry combining LTA4H with immune cell markers

  • Spatial transcriptomics to correlate LTA4H expression with immune microenvironment

  • Flow cytometry protocols for analyzing LTA4H in tumor-infiltrating lymphocytes

• Checkpoint Inhibitor Combination Studies:

  • Investigate synergistic effects between LTA4H inhibitors and checkpoint blockers

  • Develop experimental protocols for sequential vs. concurrent administration

  • Establish biomarker panels to predict combination therapy response

• Ex Vivo Tumor Modeling:

  • Organoid culture systems incorporating immune components

  • Patient-derived tumor spheroids treated with LTA4H inhibitors

  • Live-cell imaging approaches to track immune cell-tumor interactions

• Immuno-Metabolic Profiling:

  • Measure LTB4 and other eicosanoids in the tumor microenvironment

  • Correlate metabolite profiles with immune cell activation states

  • Develop mass cytometry panels to simultaneously assess LTA4H and immune activation markers

These emerging approaches will help define LTA4H's role in the complex interplay between cancer cells and the immune system, potentially leading to novel immunotherapeutic strategies targeting the LTA4H pathway.