ANGPTL4 Antibody

What is ANGPTL4 and why is it important in research?

ANGPTL4 (Angiopoietin-like protein 4) is a secreted glycoprotein with significant roles in lipid metabolism, predominantly expressed in adipose tissue and liver. Its importance stems from its function as an inhibitor of lipoprotein lipase (LPL), thereby regulating circulating triglyceride levels. Beyond metabolism, ANGPTL4 has emerged as a critical factor in tumorigenesis, angiogenesis, vascular permeability, and stem cell regulation, making it a compelling target for diverse research areas . Understanding ANGPTL4 is essential for investigating metabolic disorders, cancer progression, and vascular biology.

What applications are ANGPTL4 antibodies suitable for?

ANGPTL4 antibodies have been validated for multiple research applications with varying degrees of optimization. These include:

• Western blot (WB): Widely validated across human and animal samples

• ELISA: Particularly useful for quantitative analysis of ANGPTL4 in serum samples

• Immunohistochemistry (IHC): Effective for tissue localization studies, especially in kidney, liver and adipose tissues

• Simple Western assays: Automated capillary-based western blotting for higher throughput analysis

• Sandwich immunoassays: For highly sensitive detection of ANGPTL4 in complex samples

The application selection should be guided by your specific research question and sample type availability.

What are the typical molecular weights observed for ANGPTL4 in Western blot experiments?

The molecular weight detection for ANGPTL4 varies based on post-translational modifications and experimental conditions:

These variations reflect the glycosylation state, oligomerization, and possible proteolytic processing of ANGPTL4. When performing Western blot analysis, researchers should anticipate potential band size differences depending on tissue source and experimental conditions .

What are the optimal conditions for Western blot detection of ANGPTL4?

For successful Western blot detection of ANGPTL4, the following protocol parameters have been experimentally validated:

• Sample preparation: 30 μg of protein lysate per lane under reducing conditions

• Gel electrophoresis: 5-20% SDS-PAGE at 70V (stacking)/90V (resolving) for 2-3 hours

• Transfer conditions: 150 mA for 50-90 minutes to nitrocellulose membrane

• Blocking: 5% non-fat milk in TBS for 1.5 hours at room temperature

• Primary antibody:

  • 0.5 μg/mL of anti-ANGPTL4 (A01147) overnight at 4°C

  • 0.1 μg/mL for AF3485 antibody

  • 1:500-1:1000 dilution for 51109-1-AP antibody

• Secondary antibody: Anti-rabbit IgG-HRP at 1:5000 for 1.5 hours at room temperature

• Detection system: Enhanced chemiluminescence (ECL)

These conditions should be optimized for your specific experimental system and antibody selection.

How should I optimize immunohistochemistry protocols for ANGPTL4 detection in tissue samples?

For optimal IHC detection of ANGPTL4:

• Antigen retrieval: Use TE buffer at pH 9.0 (recommended) or citrate buffer at pH 6.0 (alternative)

• Antibody dilution: Start with 1:50-1:500 range for polyclonal antibodies like 51109-1-AP

• Tissue considerations:

  • Human, rat, and mouse kidney tissues have been positively validated

  • Placenta shows high ANGPTL4 expression and is suitable for antibody validation

  • Liver tissue from mice has shown positive detection in Western blot

• Control validation: Include appropriate negative controls and, when possible, tissues from ANGPTL4 knockout models

Note that titration experiments are essential as optimal dilutions may vary between tissue types and fixation methods .

Which experimental models are suitable for studying ANGPTL4 functions in metabolism?

Several experimental models have demonstrated utility for investigating ANGPTL4's metabolic functions:

• Adipose tissue models:

  • Human diabetic adipose samples show distinct ANGPTL4 expression patterns

  • Mouse adipocytes respond to hypoxia with altered ANGPTL4 expression

• Liver models:

  • Mouse liver tissue expresses detectable ANGPTL4 levels

  • Human hepatocellular carcinoma samples show altered expression patterns

• Genetic variants:

  • The E40K and T266M variants provide valuable models for understanding ANGPTL4 function in lipid metabolism

  • These variants show reduced plasma triglyceride levels and increased HDL-C levels

• Diabetic mouse models:

  • Useful for examining ANGPTL4's role in wound healing and vascular function

  • Allow for investigation of ANGPTL4-mediated nitric oxide production through integrin/JAK/STAT3 pathways

Each model system offers specific advantages for addressing different aspects of ANGPTL4 biology in metabolic regulation.

How can I investigate ANGPTL4's role in cancer progression and angiogenesis?

To study ANGPTL4's functions in cancer and angiogenesis, consider these methodological approaches:

• Tumor cell expression analysis:

  • Western blot analysis of ANGPTL4 in tumor samples (gastric cancer, melanoma)

  • Comparison between hypoxic and normoxic tumor regions

  • Examination of ANGPTL4 in conventional renal cell carcinoma

• Angiogenesis assays:

  • Corneal angiogenesis model for in vivo assessment

  • Miles assay for vascular permeability

  • Chicken chorioallantoic membrane assay for angiogenic response

  • Tubule formation assays with endothelial cells

• Metastasis models:

  • Melanoma to brain metastasis models, where ANGPTL4 has demonstrated promoting effects

  • Analysis of vascular activity in metastatic processes

• Hypoxia response:

  • Induction of ANGPTL4 under hypoxic conditions in endothelial cells

  • Analysis of hypoxia-responsive elements in the ANGPTL4 promoter

These approaches provide complementary insights into ANGPTL4's complex and sometimes contradictory roles in tumor biology.

How do I address inconsistent molecular weight observations for ANGPTL4 in Western blot experiments?

The variability in ANGPTL4 molecular weight (45-65 kDa) can be addressed through these analytical approaches:

• Post-translational modification analysis:

  • Treat samples with deglycosylation enzymes (PNGase F) to determine glycosylation contribution

  • Compare reducing versus non-reducing conditions to assess oligomerization states

• Domain-specific antibodies:

  • Use antibodies targeting different epitopes (N-terminal vs. C-terminal) to identify processing events

  • The A01147 antibody detects a 65 kDa band , while AF3485 detects a 59 kDa band

• Sample preparation variables:

  • Compare fresh versus frozen tissue preparations

  • Evaluate different lysis buffer compositions to preserve protein integrity

  • Test protease inhibitor cocktail formulations

• Comparative analysis:

  • Include recombinant ANGPTL4 protein standards alongside tissue samples

  • Run samples from multiple tissue sources in parallel to establish tissue-specific patterns

These approaches will help distinguish technical artifacts from biologically relevant ANGPTL4 isoforms or modifications.

How can I reconcile contradictory findings regarding ANGPTL4's role in angiogenesis?

The literature presents seemingly contradictory findings regarding ANGPTL4's pro- and anti-angiogenic properties. To address these discrepancies:

• Context-dependent analysis:

  • Examine tissue-specific effects (tumor versus normal tissue)

  • Consider the inflammatory status of the experimental model

  • Analyze acute versus chronic ANGPTL4 exposure

• Isoform-specific functions:

  • Differentiate between full-length ANGPTL4 and its cleaved fragments (N-terminal vs. C-terminal domains)

  • Determine which isoforms predominate in your experimental system

• Concentration-dependent effects:

  • Perform dose-response experiments with recombinant ANGPTL4

  • Compare physiological versus supraphysiological concentrations

• Interacting factors:

  • Investigate ANGPTL4 interactions with other angiogenic regulators (VEGF pathway)

  • Examine microenvironmental factors (hypoxia, inflammation)

  • Analyze the integrin/JAK/STAT3 signaling pathway involved in nitric oxide production

This multifaceted approach can resolve apparent contradictions by revealing the conditional nature of ANGPTL4 functions in angiogenesis.

What are the implications of ANGPTL4 as a therapeutic target in metabolic disorders?

ANGPTL4's role as a potential therapeutic target in metabolic disorders stems from several key findings:

• Genetic evidence:

  • The E40K mutation in ANGPTL4 is associated with lower plasma triglyceride levels and higher HDL-C levels

  • This mutation provides protection against coronary artery disease and metabolic disorders

• Mechanistic insights:

  • ANGPTL4 inhibits lipoprotein lipase (LPL), controlling triglyceride clearance

  • ANGPTL4 deficiency suppresses foam cell formation in macrophages and protects against atherosclerosis

• Diabetes connections:

  • Contradictory findings regarding ANGPTL4 serum levels in Type 2 Diabetes require resolution

  • ANGPTL4 accelerates wound healing in diabetic mice through angiogenesis modulation

• Targeting strategies:

  • Development of antibodies that can neutralize ANGPTL4's LPL inhibition

  • Small molecule inhibitors that disrupt ANGPTL4 oligomerization

  • Peptide mimetics of the E40K variant

Researchers investigating ANGPTL4 as a therapeutic target should consider these multiple physiological roles to anticipate potential off-target effects in clinical applications.

How can advanced imaging techniques be combined with ANGPTL4 antibodies to investigate its tissue-specific functions?

Emerging imaging approaches can enhance ANGPTL4 research through:

• Multiplex immunofluorescence:

  • Co-localization studies of ANGPTL4 with cellular markers

  • Assessment of ANGPTL4 expression in relation to hypoxic regions

  • Quantification of secreted versus cell-associated ANGPTL4

• Intravital microscopy:

  • Real-time visualization of ANGPTL4 dynamics in living tissues

  • Analysis of vascular permeability modulation in response to ANGPTL4

• Super-resolution microscopy:

  • Nanoscale localization of ANGPTL4 relative to subcellular structures

  • Visualization of ANGPTL4 interactions with receptors and extracellular matrix components

• Mass spectrometry imaging:

  • Label-free detection of ANGPTL4 in tissue sections

  • Correlation with lipid distribution patterns to connect structure with function

These advanced imaging approaches, when combined with validated ANGPTL4 antibodies, provide powerful tools for dissecting the complex biology of this multifunctional protein in intact tissues.