ANGPTL1 Antibody

What is ANGPTL1 and what biological functions does it serve?

ANGPTL1 (Angiopoietin-like protein 1) belongs to the angiopoietin-like family of proteins and functions primarily as a tumor suppressor in various cancers. It has been demonstrated to inhibit angiogenesis, tumor growth, metastasis, and invasion. ANGPTL1 has significant regulatory effects on cell migration, invasion, and cancer stemness properties . Structurally, it contains a coiled-coil domain in the N-terminus and a fibrinogen-like domain in the C-terminus, with a molecular weight of approximately 57 kDa .

Where is ANGPTL1 expressed in normal human tissue?

ANGPTL1 shows a tissue-specific expression pattern with high expression observed in the adrenal gland, placenta, thyroid gland, heart, skeletal muscle, and small intestine. Comparatively lower expression levels are found in the testis, ovary, colon, pancreas, kidney, and stomach . This tissue distribution pattern is important to consider when designing experiments or interpreting results from different tissue samples.

What signaling pathways does ANGPTL1 participate in or regulate?

ANGPTL1 interacts with several major signaling pathways that control cellular proliferation, migration, and invasion:

• Integrin α1β1/focal adhesion kinase (FAK) signaling axis

• Janus kinase (JAK)/STAT3 signaling pathway

• MAPK/ERK1/2 signaling cascade

• PI3K/Akt signaling pathway

• FOXO3a-dependent signaling that regulates SOX2 expression

In thyroid cancer, ANGPTL1 negatively regulates the activation of MAPK/ERK1/2 and PI3K/AKT signaling pathways, which are associated with cancer recurrence .

How does ANGPTL1 expression differ between cancer and normal tissues?

Multiple studies have demonstrated that ANGPTL1 is significantly downregulated in various cancer types compared to matched adjacent non-cancerous tissues. In differentiated thyroid cancer (DTC), ANGPTL1 expression levels are lower than in adjacent normal thyroid tissues, with expression decreasing progressively with cancer advancement . Similarly, in colorectal cancer (CRC), ANGPTL1 is downregulated and inversely correlated with metastasis and poor clinical outcomes . This pattern suggests a consistent tumor-suppressive role across different cancer types.

What mechanisms underlie ANGPTL1's effects on cancer cell behavior?

ANGPTL1 inhibits cancer progression through multiple mechanisms:

• Proliferation inhibition: Overexpression of ANGPTL1 in thyroid cancer cells (TPC-1) reduces cell viability, while knockdown increases proliferation .

• Migration and invasion suppression: ANGPTL1 significantly decreases the migration distance and invasive ability of cancer cells by modulating migration/invasion signaling pathways .

• Cancer stemness regulation: In colorectal cancer, ANGPTL1 suppresses cancer stem cell marker expression and sphere formation by enhancing FOXO3a expression, which reduces SOX2 expression .

• Metastasis prevention: ANGPTL1 reduces liver metastasis, tumor growth, and tumorigenicity in animal models .

How is ANGPTL1 associated with cancer recurrence and clinical outcomes?

Patients with DTC recurrence exhibit significantly lower ANGPTL1 expression compared to patients without recurrence . Gene signature analysis shows that low ANGPTL1 levels are associated with activation of MAPK/ERK1/2 and PI3K/Akt signaling pathways, which are linked to thyroid cancer recurrence . In colorectal cancer, ANGPTL1 expression is inversely correlated with metastasis and confers better clinical outcomes . These findings suggest ANGPTL1 could serve as a prognostic biomarker for cancer recurrence and survival.

What are optimal conditions for using ANGPTL1 antibodies in Western blot analysis?

For optimal Western blot results with ANGPTL1 antibodies:

• Sample preparation: Use standard protein extraction protocols with protease inhibitors to prevent degradation.

• Loading amount: Load 20-40 μg of total protein per lane for cell lysates; adjust based on ANGPTL1 expression level in your sample type.

• Gel percentage: Use 10% SDS-PAGE gels for optimal separation around the 57 kDa marker (ANGPTL1's molecular weight) .

• Antibody dilution: Follow manufacturer recommendations, typically 1:1000 for primary antibody incubation.

• Blocking: Use 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Controls: Include positive controls from tissues known to express ANGPTL1 (adrenal gland, placenta, or thyroid tissue) .

• Detection: Both chemiluminescence and fluorescence-based detection systems are suitable.

How can researchers effectively measure changes in ANGPTL1-regulated pathways?

To comprehensively assess ANGPTL1-regulated pathways:

• Phosphorylation analysis: Measure activation states of key signaling proteins (p-ERK1/2, p-AKT, p-STAT3) using phospho-specific antibodies by Western blot.

• Gene expression analysis: Perform qRT-PCR for downstream targets in MAPK/ERK and PI3K/AKT pathways.

• Transcriptome profiling: Use RNA-seq followed by Gene Set Enrichment Analysis (GSEA) to identify enriched pathway signatures, as performed in ANGPTL1 studies on thyroid cancer .

• Protein-protein interaction: Co-immunoprecipitation to detect interaction between ANGPTL1 and integrin α1β1 or other binding partners.

• Functional assays: Cell proliferation (CCK-8 assay), migration (wound healing assay), and invasion (Transwell invasion assay) to correlate pathway changes with phenotypic effects .

What cell and animal models are most appropriate for studying ANGPTL1's effects?

Cell Models:

• TPC-1 thyroid cancer cells have been successfully used to study ANGPTL1's effects on proliferation, migration, and invasion

• Colorectal cancer cell lines are suitable for investigating ANGPTL1's impact on cancer stemness

• HT1080 fibrosarcoma cells engineered to overexpress ANGPTL1 show reduced tumorigenicity

Animal Models:

• Nude mice with intravenous injection of ANGPTL1-expressing cells (e.g., HT1080-ANGPTL1) for tumorigenicity and metastasis studies

• Orthotopic implantation models for studying tissue-specific effects

• Xenograft models to assess ANGPTL1's impact on tumor growth and metastasis in vivo

What are key considerations when selecting an ANGPTL1 antibody?

When selecting an ANGPTL1 antibody, researchers should consider:

• Application compatibility: Verify the antibody is validated for your specific application (WB, IHC, IF, etc.)

• Species reactivity: Ensure reactivity with your experimental model (human, mouse, etc.)

• Epitope location: Consider whether N-terminal or C-terminal targeting is more appropriate based on potential post-translational modifications or splice variants

• Mono vs. polyclonal: Polyclonal antibodies (like DF9210) offer higher sensitivity but potentially less specificity than monoclonals

• Validation data: Review manufacturer-provided validation data and published literature using the antibody

• Citation history: Check if the antibody has been successfully used in publications with similar experimental conditions

• Lot-to-lot consistency: Consider manufacturers with good quality control practices to ensure reproducibility

How should researchers troubleshoot non-specific binding or weak signals with ANGPTL1 antibodies?

For non-specific binding:

• Increase blocking time/concentration (5-10% blocking agent)

• Optimize primary antibody dilution (try series: 1:500, 1:1000, 1:2000)

• Increase washing duration and frequency (5 washes × 5 minutes)

• Add 0.1-0.5% Tween-20 to antibody dilution buffer

• Pre-absorb antibody with non-specific proteins

• Try alternative blocking agents (switch between milk and BSA)

For weak signals:

• Increase protein loading amount

• Reduce antibody dilution

• Extend primary antibody incubation (overnight at 4°C)

• Use signal enhancement systems

• Try different sample preparation methods to improve protein extraction

• Verify ANGPTL1 expression level in your specific tissue/cell type

• Consider tissue-specific expression patterns and adjust protocol accordingly

What controls should be included when studying ANGPTL1 expression or function?

Essential controls include:

• Positive tissue controls: Samples from adrenal gland, placenta, thyroid gland, heart, or skeletal muscle known to express ANGPTL1

• Negative tissue controls: Tissues with minimal ANGPTL1 expression or cell lines with ANGPTL1 knockdown

• Loading controls: Housekeeping proteins (β-actin, GAPDH) for Western blot normalization

• Antibody controls:

  • Primary antibody omission

  • Isotype control

  • Blocking peptide competition (if available)

• Genetic manipulation controls:

  • Empty vector controls for overexpression studies

  • Non-targeting siRNA for knockdown experiments

  • Rescue experiments to confirm specificity of phenotypic effects

• Functional assay controls: Positive and negative controls specific to each functional assay being performed

How can ANGPTL1 serve as a biomarker for cancer diagnosis and prognosis?

ANGPTL1 shows significant potential as both a diagnostic and prognostic biomarker:

• Diagnostic applications: Serum ANGPTL1 levels are lower in patients with differentiated thyroid cancer compared to those with benign thyroid nodules, with an AUC of 0.696, sensitivity of 60.71%, and specificity of 80.77% in ROC analysis . This suggests potential utility as a non-invasive diagnostic biomarker.

• Prognostic applications:

  • In DTC, lower ANGPTL1 expression correlates with cancer recurrence

  • In colorectal cancer, ANGPTL1 expression inversely correlates with metastasis and poorer clinical outcomes

  • ANGPTL1 expression decreases with thyroid cancer progression, making it a potential marker for disease staging

• Predictive value: ANGPTL1 may help predict treatment response by indicating activation of specific signaling pathways (MAPK/ERK1/2, PI3K/Akt) associated with drug resistance

How should researchers address discrepancies between ANGPTL1 mRNA and protein expression?

When facing discrepancies between ANGPTL1 mRNA and protein levels:

• Consider post-transcriptional regulation: Evaluate microRNA regulation, RNA stability factors, or RNA-binding proteins that might affect translation efficiency

• Examine post-translational modifications: Investigate potential proteolytic processing, phosphorylation, or other modifications that might affect protein stability or antibody recognition

• Assess protein half-life: Determine if differences reflect variations in protein turnover rather than synthesis

• Verify detection methods: Ensure primers and antibodies target conserved regions unaffected by splice variants

• Use complementary approaches: Combine transcriptomic (RNA-seq, qRT-PCR) with proteomic methods (Western blot, mass spectrometry) and functional assays

• Spatial and temporal considerations: Consider whether samples were collected at different time points or from heterogeneous tissue regions

• Statistical validation: Apply appropriate statistical methods to determine if discrepancies are significant or within expected biological variation

What are promising research directions for ANGPTL1 in cancer therapeutics?

Future research on ANGPTL1 in cancer therapeutics could explore:

• Recombinant ANGPTL1 therapy: Investigating the antitumor effects of recombinant ANGPTL1 administration in preclinical models, building on observations of its tumor-suppressive properties

• Pathway-specific targeting: Developing therapies that activate the downstream pathways modulated by ANGPTL1, particularly FOXO3a activation or inhibition of JAK2/STAT3 signaling

• Combination therapies: Exploring synergistic effects of ANGPTL1-based therapies with existing cancer treatments, especially those targeting MAPK/ERK1/2 or PI3K/Akt pathways

• Biomarker-guided treatment: Using ANGPTL1 expression levels to guide treatment decisions and predict recurrence risk in personalized medicine approaches

• Gene therapy approaches: Developing vectors for localized ANGPTL1 expression in tumors to inhibit progression and metastasis

• Cancer stemness targeting: Leveraging ANGPTL1's ability to reduce cancer stem cell properties to overcome therapy resistance and prevent recurrence

• Metastasis prevention: Focusing on ANGPTL1's anti-migratory and anti-invasive properties to develop adjuvant therapies specifically targeting metastatic spread