LTA4H Antibody

What is LTA4H and why is it significant for antibody-based research?

LTA4H (Leukotriene A4 Hydrolase) is a bifunctional zinc metalloenzyme that comprises both epoxide hydrolase (EH) and aminopeptidase activities. Its significance stems from:

• Acting as an epoxide hydrolase to catalyze the conversion of LTA4 to the pro-inflammatory mediator leukotriene B4 (LTB4)

• Possessing aminopeptidase activity, with high affinity for N-terminal arginines of various synthetic tripeptides

• Counteracting inflammation through its aminopeptidase activity by cleaving the neutrophil attractant Pro-Gly-Pro (PGP)

• Being involved in the biosynthesis of resolvin E1 and 18S-resolvin E1 from eicosapentaenoic acid, which show potent anti-inflammatory properties

In research contexts, LTA4H antibodies are valuable tools for studying inflammatory processes, cancer biology, and neurological conditions where LTA4H plays a significant role.

Which applications are most suitable for LTA4H antibody detection?

Based on validated research data, LTA4H antibodies can be successfully employed in multiple applications:

When selecting an application, consider your specific research question, sample type, and downstream analyses. For protein quantification, WB is preferred, while tissue localization studies benefit from IHC approaches .

What tissue and cell types show significant LTA4H expression?

Understanding expression patterns is crucial for experimental design. LTA4H expression has been documented in:

Human tissues: Skin, brain, heart, kidney, lung, ovary, placenta, spleen, and testis show positive immunohistochemical detection . Additionally, LTA4H is overexpressed in actinic keratosis (AK) and squamous cell carcinoma (SCC) compared to normal skin tissues .

Cell lines: Positive Western blot detection has been confirmed in:

• HEK-293 cells (human embryonic kidney)

• Jurkat cells (human T lymphocytes)

• NTera-2 cells (human testicular embryonic carcinoma)

• NCI-N87 cells (human gastric carcinoma)

• C2C12 cells (mouse myoblast)

Neuronal expression: Recent research has revealed that LTA4H protein is detectable in brain cortex homogenates and colocalizes with the nuclear marker NeuN throughout the brain, indicating neuronal expression .

How should I validate LTA4H antibody specificity for my research?

Antibody validation is critical for ensuring reliable results. A comprehensive validation approach includes:

• Knockout/knockdown controls: Use LTA4H knockout cell lines (e.g., Human LTA4H knockout HEK-293T cell line) as negative controls in Western blot to confirm specificity .

• Multiple antibody comparison: Test at least two antibodies targeting different epitopes of LTA4H. For example, compare results between polyclonal antibodies (e.g., ab23677) and monoclonal antibodies (e.g., EPR5712 clone, ab109434) .

• Cross-reactivity assessment: Verify species reactivity claims. Many LTA4H antibodies show reactivity with human, mouse, and rat samples, but this should be experimentally confirmed in your specific samples .

• Expected molecular weight verification: Confirm band detection at the expected molecular weight (typically 69 kDa), although observed weights may range from 57-70 kDa depending on post-translational modifications .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm signal abrogation in your specific application.

What are the optimal sample preparation methods for LTA4H detection?

Sample preparation significantly impacts antibody detection quality:

For Western Blot:

• Use RIPA buffer supplemented with protease inhibitors

• For cell samples, lyse 1-3×10⁶ cells in 100-300 μL buffer

• For tissue samples, homogenize 50-100 mg tissue in 500-1000 μL buffer

• Clarify lysates by centrifugation at 14,000×g for 15 minutes at 4°C

• Load 20-30 μg of total protein per lane

For Immunohistochemistry:

• Fix tissues in 10% neutral buffered formalin

• Perform heat-mediated antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0)

• Optimal section thickness: 4-6 μm

• Block endogenous peroxidase activity with 3% H₂O₂

For Immunoprecipitation:

• Use gentler lysis buffers (e.g., NP-40 buffer) to preserve protein-protein interactions

• Pre-clear lysates with protein A/G beads

• Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

What controls should be included in LTA4H antibody experiments?

Rigorous experimental design requires appropriate controls:

Positive controls:

• Cell lines with known LTA4H expression (HEK-293, Jurkat, A431, SCC12, SCC13, NTera-2, NCI-N87)

• Tissues with confirmed expression (skin, brain, heart, kidney, lung)

Negative controls:

• LTA4H knockout cell lines (e.g., ab266467)

• Primary antibody omission control

• Isotype control antibody

• Tissues known to lack expression (some LTA4H antibodies do not detect expression in basal cell carcinoma)

Loading/normalization controls:

• For WB: GAPDH (ab8245) or β-actin

• For IHC: Adjacent serial sections stained with isotype control

How can LTA4H antibodies be used to investigate its dual enzymatic functions?

LTA4H possesses both epoxide hydrolase and aminopeptidase activities, which can be studied using antibody-based approaches:

• Activity-based immunoprecipitation: Immunoprecipitate LTA4H from cellular lysates using antibodies like ab109434 or 13662-1-AP, then measure:

  • Epoxide hydrolase activity using LTA4 substrate and quantifying LTB4 production by ELISA

  • Aminopeptidase activity using synthetic tripeptide substrates like Pro-Gly-Pro (PGP)

• Subcellular localization studies: The differential localization of LTA4H can provide insights into its dual functions:

  • Intracellular LTA4H (cytosolic) is associated with epoxide hydrolase activity

  • Extracellular LTA4H correlates with aminopeptidase activity

  • Use immunofluorescence with antibodies like EPR5713 (ab133512) to determine subcellular distribution

• Cigarette smoke inhibition analysis: Research shows cigarette smoke selectively inhibits LTA4H aminopeptidase activity while preserving epoxide hydrolase activity:

  • Treat cells with cigarette smoke extract

  • Immunoprecipitate LTA4H

  • Assess both enzymatic activities to measure differential inhibition

What approaches can resolve contradictory LTA4H antibody data in cancer research?

Cancer research using LTA4H antibodies sometimes yields contradictory results. To resolve these discrepancies:

• Cell type-specific expression analysis: LTA4H overexpression varies by cancer type:

  • Overexpressed in skin squamous cell carcinoma (SCC), lung and colon cancer cell lines

  • Not overexpressed in normal skin cells, malignant melanoma cells, or basal cell carcinoma

  Perform systematic profiling across multiple cell lines using consistent antibody concentrations.

• Clinical correlation studies: Correlate LTA4H expression with clinical parameters:

  • Actinic keratosis (AK) and SCC show higher LTA4H expression than normal skin

  • In LTA4H knockout mice, tumor formation is significantly reduced in the DMBA/TPA two-stage skin carcinogenesis model

  Document patient histories, tumor grades, and treatment responses alongside expression data.

• Functional validation: Complement antibody detection with functional studies:

  • siRNA or shRNA knockdown of LTA4H in cancer cells

  • Pharmacological inhibition using LTA4H inhibitors

  • Measurement of downstream effects on cell cycle, particularly G0/G1 phase arrest and p27 expression

How can LTA4H antibodies advance our understanding of neuroinflammation and cognitive function?

Recent research has revealed unexpected roles for LTA4H in neuronal function:

• Neuronal expression mapping: Using IHC with anti-LTA4H antibodies:

  • LTA4H colocalizes with NeuN (neuronal nuclear marker) throughout the brain

  • Approximately 10% of hippocampal neurons express high levels of the Lta4h gene

  • Nuclear localization in neurons may be important for coordinating LTB4 processing

• Cognitive function assessment: LTA4H inhibition has been shown to improve age-related cognitive function:

  • Measure LTA4H levels in plasma across age from healthy donors and patients with Alzheimer's Disease (AD)

  • Correlate LTA4H levels with cognitive performance metrics

  • LTA4H plasma levels correlate with plasma tau levels, a biomarker for AD

• Single-cell transcriptomics correlation: Following LTA4H inhibitor treatment:

  • Collect hippocampal nuclei and enrich for neuronal nuclei using FACS

  • Perform single-nucleus RNA sequencing

  • Identify differentially expressed genes related to synaptic organization, structure, and activity

  • Correlate protein-level changes (using antibodies) with transcriptomic alterations

What factors might affect the observed molecular weight of LTA4H in Western blots?

Researchers frequently observe variability in LTA4H molecular weight detection:

To address molecular weight variations:

• Use freshly prepared samples with protease inhibitors

• Include reducing agents in sample buffer

• Run a gradient gel (4-20%) to better resolve proteins

• Consider native versus denatured conditions for certain applications

• Verify antibody specificity using knockout controls

How can I resolve non-specific binding issues with LTA4H antibodies?

Non-specific binding is a common challenge. Address it through:

• Optimization of blocking conditions:

  • Test different blocking agents (5% BSA, 5% non-fat milk, commercial blockers)

  • Extend blocking time to 1-2 hours at room temperature

  • For phospho-specific detection, BSA is preferable to milk

• Antibody titration:

  • For WB: Test dilutions from 1:500 to 1:2000

  • For IHC: Test dilutions from 1:20 to 1:200

  • Document each condition with appropriate controls

• Buffer optimization:

  • Add 0.1-0.5% Tween-20 to wash buffers

  • Consider adding 0.1% Triton X-100 to antibody diluent

  • For high background in tissues, include 0.3% hydrogen peroxide in methanol to block endogenous peroxidases

• Pre-adsorption:

  • Pre-incubate antibody with IgG-depleted tissue lysates from species of secondary antibody

  • Use protein A/G beads to remove non-specific binding components

What are the most reliable quantification methods for LTA4H expression analysis?

Accurate quantification is essential for comparative studies:

• Western blot densitometry:

  • Use linear range of detection (avoid saturated signals)

  • Normalize to housekeeping proteins (GAPDH, β-actin)

  • Include a standard curve of recombinant LTA4H protein

  • Use at least three biological replicates

• IHC scoring systems:

  • H-score (combines intensity and percentage of positive cells)

  • Automated image analysis with positive pixel algorithms

  • Tissue microarrays for high-throughput analysis

• Flow cytometry quantification:

  • Mean fluorescence intensity (MFI)

  • Comparison to isotype controls

  • Use of calibration beads for absolute quantification

• Mass spectrometry validation:

  • Selected reaction monitoring (SRM) for absolute quantification

  • Compare antibody-based quantification with MS-based approaches

  • Identify and quantify specific post-translational modifications

How can LTA4H antibodies be used to study the enzyme's role in inflammation resolution?

Beyond its pro-inflammatory functions, LTA4H plays a role in inflammation resolution:

• Dual-function analysis: LTA4H's aminopeptidase activity cleaves and inactivates the neutrophil chemoattractant Pro-Gly-Pro (PGP):

  • Use antibodies to immunoprecipitate LTA4H from biological fluids

  • Measure aminopeptidase activity against PGP substrate

  • Compare activity in inflammatory versus resolving conditions

• Resolution mediator studies: LTA4H is involved in the biosynthesis of resolvin E1:

  • Investigate LTA4H expression timing during inflammation progression and resolution

  • Correlate with resolvin E1 levels

  • Use antibodies for immunoprecipitation followed by activity assays

• Cigarette smoke effect characterization: Cigarette smoke selectively inhibits LTA4H's aminopeptidase activity:

  • Expose cells/tissues to cigarette smoke extract

  • Assess changes in LTA4H expression and subcellular localization using antibodies

  • Correlate with PGP accumulation and neutrophil persistence

What novel applications of LTA4H antibodies are emerging in neurodegenerative disease research?

Recent studies reveal promising applications in neurodegenerative research:

• Cognitive function biomarker development:

  • LTA4H plasma levels correlate with cognitive impairment in human patients

  • LTA4H plasma levels also correlate with plasma tau levels, a biomarker for Alzheimer's Disease

  • Use antibodies for developing standardized LTA4H quantification assays

• Neuronal LTA4H signaling pathway elucidation:

  • LTA4H inhibition improves synaptic function in aged mice

  • Investigate LTA4H expression in different neuronal populations

  • Use antibodies for co-immunoprecipitation to identify neuronal-specific LTA4H interaction partners

• Blood-brain barrier studies:

  • LTA4H may affect brain capillary health and neurovascular unit function

  • Use antibodies to study LTA4H expression in brain endothelial cells

  • Investigate changes under pathological conditions