NCEH1 Antibody

What is NCEH1 and why is it important in research?

NCEH1 (Neutral Cholesterol Ester Hydrolase 1) is a protein involved in lipid metabolism and protein dephosphorylation. In humans, the canonical protein has 408 amino acid residues with a molecular mass of 45.8 kDa and is primarily localized in the cell membrane . NCEH1 belongs to the 'GDXG' lipolytic enzyme protein family and undergoes post-translational modifications including N-glycosylation .

The protein is particularly important in research due to its:

• Expression in monocyte-derived macrophages

• Role in cholesterol metabolism and potential implications for atherosclerosis

• Emerging role as a biomarker for certain cancers including gastric cancer

• Function in endothelial cells related to diabetes complications

Alternative names for this target include acetylalkylglycerol acetylhydrolase, alkylacetylglycerol acetylhydrolase, arylacetamide deacetylase-like 1, and 2-acetyl MAGE hydrolase .

What applications are NCEH1 antibodies commonly used for?

NCEH1 antibodies are versatile research tools with multiple validated applications:

Most commercially available NCEH1 antibodies are validated for Western Blotting, ELISA, and IHC applications, with some also validated for IF, FCM, and IP . When selecting an antibody, researchers should verify which applications have been validated for their specific experimental needs.

How do I select the appropriate NCEH1 antibody for my experiment?

When selecting an NCEH1 antibody, consider these critical factors:

• Target species reactivity: Ensure the antibody recognizes NCEH1 in your experimental species. Available antibodies show reactivity to various species including human, mouse, rat, cow, dog, horse, guinea pig, rabbit, and zebrafish .

• Antibody type: Choose between:

  • Polyclonal antibodies: Recognize multiple epitopes (most NCEH1 antibodies are polyclonal)

  • Monoclonal antibodies: Recognize a single epitope (higher specificity)

• Target region: Select antibodies targeting specific regions based on your research needs:

  • N-terminal region antibodies

  • C-terminal region antibodies

  • Middle region antibodies

  • Full-length protein antibodies

• Validated applications: Verify the antibody has been validated for your specific application (WB, ELISA, IHC, etc.)

• Host species: Consider the host species (typically rabbit or goat for NCEH1 antibodies) to avoid cross-reactivity issues in multi-labeling experiments .

How can I validate the specificity of my NCEH1 antibody?

Validating antibody specificity is crucial for reliable research. For NCEH1 antibodies, employ these methodologies:

• Positive and negative controls:

  • Positive: Use tissue/cells known to express NCEH1 (e.g., monocyte-derived macrophages)

  • Negative: Use NCEH1 knockout models or NCEH1-silenced cells via siRNA/shRNA

• Peptide competition assay: Pre-incubate your antibody with the immunizing peptide before application to your sample. Signal disappearance indicates specificity.

• Knockout validation: Compare results between wild-type and NCEH1 knockout samples. A specific antibody will show no signal in knockout samples, as demonstrated in Figure 4F of Kratky's report where Western blotting with a specific anti-NCEH1 antibody showed complete absence of signal in Nceh1 knockout mouse macrophages .

• Multiple antibody comparison: Use antibodies targeting different NCEH1 epitopes and compare detection patterns.

• Molecular weight verification: Confirm the detected band is at the expected molecular weight (~45.8 kDa for human NCEH1).

• Antibody titration: Optimize antibody concentration to maximize signal-to-noise ratio.

What are the best practices for using NCEH1 antibodies in Western blotting?

For optimal Western blotting results with NCEH1 antibodies:

• Sample preparation:

  • Use appropriate lysis buffers with protease inhibitors

  • For membrane-associated NCEH1, ensure proper membrane protein extraction

  • Include positive controls (monocyte-derived macrophages express NCEH1)

• Electrophoresis conditions:

  • Use 10-12% SDS-PAGE gels for optimal separation around 45.8 kDa

  • Run at 100-120V to prevent protein degradation

• Transfer optimization:

  • Semi-dry or wet transfer at 100V for 60-90 minutes or 30V overnight

  • Use PVDF membranes for better protein retention

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST

  • Typical dilutions for primary NCEH1 antibodies range from 1:500 to 1:2000

  • Incubate primary antibody overnight at 4°C for best results

• Detection troubleshooting:

  • If background is high, increase blocking time or washing steps

  • If signal is weak, increase antibody concentration or incubation time

  • Consider enhanced chemiluminescence (ECL) for sensitive detection

• Expected results:

  • Human NCEH1: ~45.8 kDa band

  • Consider possible post-translational modifications (e.g., N-glycosylation) may affect migration pattern

  • In tunicamycin-treated samples, expect altered band pattern due to inhibition of N-glycosylation

How can I optimize immunohistochemistry protocols using NCEH1 antibodies?

For successful IHC with NCEH1 antibodies:

• Sample preparation:

  • For paraffin sections: Use 10% neutral buffered formalin fixation

  • For frozen sections: Flash freeze tissue and maintain at -80°C

• Antigen retrieval methods:

  • Heat-induced epitope retrieval: Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Enzymatic retrieval: Proteinase K (1-10 μg/ml) for 10-20 minutes at 37°C

• Antibody optimization:

  • Determine optimal dilution range (typically 1:100-1:500)

  • Incubate at 4°C overnight for best sensitivity

  • Use biotin-streptavidin amplification systems for enhanced sensitivity

• Controls to include:

  • Positive tissue control (gastric cancer tissue shows elevated NCEH1 expression)

  • Negative control (omit primary antibody)

  • Isotype control (use non-specific IgG)

• Specific considerations:

  • For detecting NCEH1 in macrophages, consider double staining with macrophage markers

  • For gastric cancer diagnosis, optimize protocols for sensitivity of 77.5% and specificity of 73.6%

How should I interpret conflicting data regarding NCEH1 function in macrophage cholesterol metabolism?

The scientific literature reveals a significant controversy regarding NCEH1's role in macrophage cholesterol metabolism:

• Conflicting experimental evidence:

  • Igarashi et al. reported that NCEH1 knockdown reduced neutral cholesterol ester hydrolase activity by ~50% in human macrophages, concluding it is "quantitatively the most important neutral cholesterol ester hydrolase in human macrophages" .

  • Kratky et al. demonstrated identical neutral cholesterol ester hydrolase activity in wild-type and Nceh1 knockout mice, arguing against a critical role for NCEH1 .

• Methodological considerations:

  • Knockdown vs. knockout approaches: Knockdown experiments may retain residual protein activity

  • Complete vs. partial silencing: CES1 knockdown in Igarashi's work was less efficient, leaving substantial amounts of CES1 protein

  • Compensatory mechanisms: Gene knockouts may trigger compensatory upregulation of other hydrolases

• Reconciling contradictions:

  • Species differences: Human and mouse macrophages may utilize different enzymes

  • Redundant systems: Multiple hydrolases may perform similar functions

  • Context-dependent activity: NCEH1's importance may vary with cellular conditions

• Experimental design recommendations:

  • Use multiple approaches (knockout, knockdown, inhibitors)

  • Employ both human and mouse models

  • Measure enzyme activity and lipid composition

  • Assess multiple candidate hydrolases simultaneously

  • Consider compensatory mechanisms

When designing experiments to investigate NCEH1 function, incorporate controls addressing these contradictions and interpret results in light of this ongoing scientific debate .

What are the current approaches for using NCEH1 as a biomarker in gastric cancer research?

NCEH1 shows promise as a gastric cancer biomarker based on recent proteomics research:

• Diagnostic performance metrics:

  • Sensitivity: 77.5% for gastric cancer detection

  • Specificity: 73.6% compared to adjacent tissues and benign controls

  • Area Under ROC Curve (AUC): 0.792

• Improved performance with combined biomarkers:

  • NCEH1 + NSDHL (NAD(P)-dependent steroid dehydrogenase-like):

    • Combined sensitivity: 85.7%

    • Combined specificity: 83%

    • Combined AUC: 0.872

• Clinical correlations:

  • NCEH1 overexpression significantly associated with:

    • Tumor histological classification

    • Local invasion patterns

  • Combined NCEH1/NSDHL analysis correlates with:

    • Histological grade

    • TNM II-IV staging

• Methodological approaches:

  • Tissue-based detection: IHC validation in 129 paired gastric cancer and adjacent tissues

  • Quantitative proteomics: iTRAQ-labeled quantitative proteomics for biomarker identification

  • Validation cohorts: Must include adjacent tissues and benign healthy controls

• Research design considerations:

  • Include sufficient sample sizes for statistical power

  • Ensure appropriate control tissues (adjacent normal and true normal)

  • Consider combining with other biomarkers for improved sensitivity/specificity

  • Validate across different patient populations

What are the latest techniques for investigating NCEH1 function in endothelial cells under diabetic conditions?

Recent research has revealed NCEH1's role in endothelial function during diabetes, with several advanced methodological approaches:

• Genetic manipulation strategies:

  • Endothelial-specific knockdown/overexpression using AAV5 vectors

  • TIE1 promoter-controlled expression for endothelial specificity

  • Viral genome particle delivery (8-11 × 10^11 viral genome particles per mouse)

• Functional assessment methods:

  • Endothelium-dependent relaxation (EDR) measurement in isolated aortic rings

  • Ex vivo exposure of mouse aortae to high glucose conditions

  • Primary endothelial cell isolation and culture under diabetic conditions

• Molecular interaction studies:

  • NCEH1 interaction with E3 ubiquitin-protein ligase ZNRF1

  • Caveolin-1 (Cav-1) degradation through ubiquitination pathway

  • Dissociation of endothelial nitric oxide synthase (eNOS) from Cav-1

• Protein stability assessment:

  • Cycloheximide (CHX) chase assay (100 μg/mL)

  • Time course: 0, 3, 6, 9, 12, 15, 18, 24 hours

  • Immunoblotting for protein degradation analysis

• Gene expression analysis:

  • qRT-PCR with specific primers:

    • NCEH1: 5′-AAGGTCTTCTCCGAAAGTGAAGG-3′ (Forward)
      5′-CCTCCGTGGATATAGATGACGC-3′ (Reverse)

    • Cav-1: 5′-GCGACCCCAAGCATCTCAA-3′ (Forward)
      5′-ATGCCGTCGAAACTGTGTGT-3′ (Reverse)

• In vivo diabetes models:

  • High-fat diet (HFD)-induced diabetic mice

  • Assessment of NCEH1 expression and activity in diabetic aortae

  • Comparison of wild-type versus NCEH1-deficient mice under diabetic conditions

This methodological framework provides a comprehensive approach to investigating NCEH1 function in the context of diabetic endothelial dysfunction.

How can I design effective knockdown/knockout experiments for NCEH1 functional studies?

For robust NCEH1 functional studies, consider these experimental design principles:

• Gene silencing approaches:

  • siRNA transfection: Transient knockdown, suitable for short-term studies

  • shRNA via adenoviral vectors: Longer-term silencing as used by Igarashi et al.

  • CRISPR-Cas9: Complete knockout, consider using Tie1-driven Cas9 for endothelial-specific deletion

• Critical controls:

  • Scrambled/non-targeting RNA controls

  • Empty vector controls for viral transduction

  • Wild-type isogenic controls for knockout models

  • Rescue experiments (re-expression of NCEH1) to confirm specificity

• Validation methods:

  • Protein levels: Western blot with specific antibodies

  • Enzyme activity: Neutral cholesterol ester hydrolase activity assays

• Functional readouts:

  • Lipid metabolism: Cholesterol ester hydrolysis, lipid droplet quantification

  • Endothelial function: NO production, vasodilation, eNOS activity

  • Cell signaling: Caveolin-1 interaction, ubiquitination pathways

• Common pitfalls and solutions:

  • Incomplete knockdown: Optimize transfection conditions, use multiple siRNAs

  • Off-target effects: Use multiple silencing sequences, validate with rescue experiments

  • Compensatory mechanisms: Analyze expression of related hydrolases

  • Cell type specificity: Use tissue-specific promoters (e.g., TIE1 for endothelial cells)

What are the best approaches for analyzing NCEH1 post-translational modifications?

NCEH1 undergoes important post-translational modifications that affect its function. Here are methodological approaches to study these modifications:

• N-glycosylation analysis:

  • Enzymatic deglycosylation: PNGase F treatment followed by Western blot

  • Glycosylation inhibition: Tunicamycin treatment (5 μg/ml) to prevent N-glycosylation

  • Mobility shift assays: Compare migration patterns before/after deglycosylation

  • Site-directed mutagenesis: Mutate predicted N-glycosylation sites (Asn-X-Ser/Thr)

• Phosphorylation studies:

  • Phospho-specific antibodies: For known phosphorylation sites

  • Phosphatase treatment: Compare with/without phosphatase treatment

  • Mass spectrometry: Identify phosphorylation sites

  • Kinase inhibitors: Determine regulatory kinases

• Ubiquitination analysis:

  • Co-immunoprecipitation with ZNRF1 (E3 ubiquitin ligase)

  • Ubiquitin pull-down assays

  • Proteasome inhibitors: MG132 treatment to accumulate ubiquitinated proteins

  • Cycloheximide chase assays: Monitor protein degradation rates

• Protein-protein interaction mapping:

  • Co-immunoprecipitation: NCEH1 interaction with Cav-1 and ZNRF1

  • Proximity ligation assay: In situ visualization of protein interactions

  • FRET/BRET: Real-time interaction monitoring in live cells

  • Mass spectrometry: Identify interacting partners

• Localization studies:

  • Subcellular fractionation: Membrane vs. cytosolic fractions

  • Immunofluorescence: Co-localization with organelle markers

  • Live cell imaging: Trafficking of fluorescently tagged NCEH1

How can contradictory results in NCEH1 research be explained by methodological differences?

Scientific controversies in NCEH1 research can often be traced to methodological differences:

• Knockout vs. knockdown approaches:

  • Knockout: Complete gene deletion with potential compensatory mechanisms

  • Knockdown: Partial reduction (typically 50-95%) with remaining activity

  • Example: While Kratky et al. found identical neutral cholesterol ester hydrolase activity in Nceh1 knockout mice, Igarashi's knockdown showed 50% reduction in activity

• Species differences:

  • Human cells: NCEH1 may play a more dominant role

  • Mouse models: Other enzymes may compensate for NCEH1 loss

  • Experimental design should account for species-specific differences

• Cell type variations:

  • Primary cells vs. cell lines

  • Tissue-specific expression patterns

  • Differentiation state (e.g., monocytes vs. differentiated macrophages)

• Assay sensitivity and specificity:

  • Enzyme activity assays: Substrate specificity affects results

  • Antibody detection: Epitope accessibility in different experimental conditions

  • Example: Western blotting validation of knockout models is critical, as highlighted in the controversy between Kratky and Igarashi

• Contextual factors:

  • Lipid loading conditions

  • Inflammatory status of cells

  • Metabolic state (normal vs. disease models)

  • Example: NCEH1 function may differ between normal conditions and disease states like diabetes

• Analytical framework:

  • Statistical approaches

  • Data normalization methods

  • Interpretation of partial effects (e.g., is 50% reduction biologically significant?)

When evaluating contradictory results, researchers should carefully consider these methodological differences and design studies that directly address the specific contradictions in the literature.

What are emerging applications of NCEH1 antibodies in translational research?

NCEH1 antibodies are finding novel applications in translational research:

• Diagnostic biomarker development:

  • Gastric cancer detection: Building on the 77.5% sensitivity and 73.6% specificity

  • Multi-marker panels: Combining NCEH1 with NSDHL for improved diagnostic accuracy (85.7% sensitivity, 83% specificity)

  • Potential applications in other cancers and metabolic diseases

• Therapeutic target validation:

  • Atherosclerosis intervention: Macrophage NCEH1 as a potential drug target

  • Endothelial dysfunction in diabetes: NCEH1 as a protective factor

  • Anti-cancer approaches: Exploiting NCEH1 overexpression in tumor cells

• Patient stratification:

  • Correlation with histological classification and local invasion in cancer

  • Potential prognostic indicators

  • Treatment response prediction

• Imaging applications:

  • Immunohistochemical assessment of tumor margins

  • Potential for developing imaging agents targeting NCEH1-expressing tissues

  • Monitoring therapy response

• Molecular pathway mapping:

  • NCEH1-ZNRF1-Cav-1-eNOS pathway in endothelial function

  • Lipid metabolism networks in macrophages

  • Novel interaction partners and signaling pathways

Researchers should focus on rigorous validation of these emerging applications with appropriate controls and reproducible methodologies.

How might single-cell analysis techniques advance our understanding of NCEH1 function?

Single-cell technologies offer new opportunities to resolve controversies and advance NCEH1 research:

• Single-cell RNA sequencing applications:

  • Heterogeneity analysis: Identify subpopulations with variable NCEH1 expression

  • Temporal dynamics: Capture expression changes during cell differentiation or activation

  • Co-expression networks: Identify genes co-regulated with NCEH1

  • Compensatory mechanisms: Detect upregulation of alternative hydrolases in NCEH1-deficient cells

• Single-cell proteomics approaches:

  • Protein co-localization: NCEH1 with interaction partners like ZNRF1 and Cav-1

  • Post-translational modification patterns at single-cell level

  • Enzyme activity in individual cells

• Spatial transcriptomics/proteomics:

  • Tissue microenvironment effects on NCEH1 expression

  • Spatial relationship between NCEH1-expressing cells in diseased tissues

  • In situ visualization of NCEH1 activity

• Multi-omics integration:

  • Correlate NCEH1 expression with lipidome changes

  • Link genotype to NCEH1 expression patterns

  • Connect NCEH1 activity to metabolic profiles

• Advanced microscopy techniques:

  • Super-resolution imaging of NCEH1 subcellular localization

  • Live-cell imaging of NCEH1 trafficking

  • FRET/BRET sensors for real-time activity monitoring

These approaches could help resolve the controversy regarding NCEH1's role in cholesterol metabolism by providing cell-specific, high-resolution data on its function in different contexts.