ANGPTL4 Antibody, HRP conjugated

What is ANGPTL4 and what are its primary biological functions?

ANGPTL4 (Angiopoietin-Like Protein 4) is a secreted glycoprotein that functions as a critical regulator of lipid metabolism. Its primary biological functions include:

• Mediating inactivation of lipoprotein lipase (LPL), thus regulating triglyceride clearance from blood serum

• Playing a significant role in lipid metabolism through its inhibitory effect on LPL activity

• Potentially regulating glucose homeostasis and insulin sensitivity

• Inhibiting proliferation, migration, and tubule formation of endothelial cells

• Reducing vascular leakage and affecting endothelial cell adhesion to the extracellular matrix

• Potentially modulating tumor-related angiogenesis in certain contexts

The protein has a molecular weight of approximately 45.2 kDa, though it is often detected at around 65 kDa in Western blot applications due to post-translational modifications .

What is the structural organization of ANGPTL4 protein?

ANGPTL4 is a two-domain protein with distinct structural and functional components:

• N-terminal domain: A coiled-coil structure that mediates oligomerization of the protein and contains the LPL inhibitory activity. This domain is responsible for ANGPTL4's ability to inhibit LPL and regulate lipid metabolism .

• C-terminal domain: A fibrinogen-like domain with distinct biological functions from the N-terminal region .

After secretion, these domains can be cleaved apart by pro-protein convertases. Importantly, this cleavage enhances the inhibitory effect of the N-terminal domain on LPL activity . When designing experiments with ANGPTL4 antibodies, researchers should consider whether their antibody targets the full-length protein, the N-terminal domain, or the C-terminal domain, as this will affect experimental outcomes and interpretation.

How do HRP-conjugated ANGPTL4 antibodies work in detection systems?

HRP-conjugated ANGPTL4 antibodies function through enzymatic amplification of detection signals in immunoassays. The process follows these methodological steps:

• Primary binding: The ANGPTL4 antibody portion binds specifically to ANGPTL4 protein in the sample.

• Signal generation: The conjugated HRP enzyme catalyzes the oxidation of substrate molecules (such as TMB or DAB) in the presence of hydrogen peroxide.

• Visualization: This enzymatic reaction produces a colored, fluorescent, or chemiluminescent product that can be measured to quantify ANGPTL4 levels.

In sandwich ELISA configurations, the system typically employs:

• A capture antibody pre-coated on a microplate surface

• The sample containing ANGPTL4

• A biotinylated detection antibody specific for ANGPTL4

• Avidin-HRP conjugate that binds to the biotin molecule

The substrate solution is then added, and only wells containing the complete sandwich complex will develop color. The optical density measured at 450 nm is proportional to the ANGPTL4 concentration in the sample .

What are the optimal conditions for using HRP-conjugated ANGPTL4 antibodies in Western blot applications?

Optimizing Western blot protocols for ANGPTL4 detection requires careful attention to several parameters. Based on experimental validation data, the following methodology is recommended:

Sample Preparation:

• Load 30 μg of protein lysate per lane under reducing conditions

• ANGPTL4 can be detected in various human tissues including placenta and hepatocellular carcinoma

Electrophoresis Conditions:

• Use a 5-20% gradient SDS-PAGE gel

• Run at 70V for stacking gel and 90V for resolving gel

• Continue electrophoresis for 2-3 hours for optimal separation

Transfer Protocol:

• Transfer proteins to nitrocellulose membrane at 150 mA for 50-90 minutes

Blocking and Antibody Incubation:

• Block membrane with 5% non-fat milk/TBS for 1.5 hours at room temperature

• Incubate with primary ANGPTL4 antibody at 0.5 μg/mL overnight at 4°C

• Wash with TBS-0.1% Tween three times, 5 minutes each

• Incubate with HRP-conjugated secondary antibody at 1:5000 dilution for 1.5 hours at room temperature

Detection:

• Develop using enhanced chemiluminescence detection system

• Expected band size for ANGPTL4 is approximately 45 kDa, though it often appears at approximately 65 kDa due to post-translational modifications

These conditions have been experimentally validated to produce specific detection with minimal background signal.

How can researchers optimize ANGPTL4 detection in immunohistochemistry using HRP-conjugated systems?

Successful immunohistochemical detection of ANGPTL4 in tissue sections requires specific optimization steps:

Antigen Retrieval Protocol:

• Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) is recommended for optimal epitope exposure

• Complete retrieval is critical for consistent and reproducible results

Blocking Procedure:

• Block tissue sections with 10% goat serum to minimize nonspecific binding

• Ensure complete coverage of the tissue section

Antibody Concentration and Incubation:

• Use ANGPTL4 primary antibody at 2 μg/ml concentration

• Incubate overnight at 4°C to ensure complete antigen binding

Secondary Detection System:

• Apply peroxidase-conjugated secondary antibody specific to the primary antibody host species

• Incubate for 30 minutes at 37°C

• Visualization using DAB (3,3'-diaminobenzidine) as the chromogen

This methodology has been successfully employed in detecting ANGPTL4 in various human tissues including placenta and spleen, with high specificity and minimal background staining .

What are the mechanisms of ANGPTL4 inhibition of lipoprotein lipase and how can this interaction be studied?

ANGPTL4 inhibits lipoprotein lipase (LPL) through a reversible, noncompetitive inhibition mechanism, rather than through the previously proposed "unfolding molecular chaperone" model. To study this interaction:

Experimental Approach to Study ANGPTL4-LPL Interaction:

• Co-immunoprecipitation assays to demonstrate ANGPTL4-LPL complex formation

• Activity recovery assays showing that LPL regains activity after dissociation from ANGPTL4

• Kinetic studies to determine the noncompetitive nature of inhibition

Key Mechanistic Findings:

• LPL inhibited by ANGPTL4 exists in a complex with ANGPTL4

• Upon dissociation from ANGPTL4, LPL can regain its lipase activity

• The inhibition is noncompetitive, not involving the catalytic conversion of LPL dimers to inactive monomers

Methodological Considerations:

• Researchers can use divalent cation-dependent variants of ANGPTL4 to create regulatable systems for studying this interaction

• Chelation treatment can reverse the inhibition in these systems, confirming the reversible nature of the inhibition

This mechanistic understanding is crucial for research aimed at developing therapeutic strategies targeting hypertriglyceridemia and related disorders.

How should researchers prepare working solutions of HRP conjugates for optimal ANGPTL4 detection?

Proper preparation of HRP conjugate working solutions is critical for sensitive and reproducible ANGPTL4 detection, particularly in ELISA applications. The following methodological approach ensures optimal reagent performance:

HRP Conjugate Preparation Protocol:

• Calculate the required volume based on the experimental design (100 μL/well)

• Always prepare slightly more than calculated to account for pipetting errors

• Centrifuge the concentrated HRP conjugate at 800×g for 1 minute to collect all liquid

• Dilute the 100× concentrated HRP conjugate to 1× working solution using appropriate diluent

• Use precise dilution ratio of 1:99 (Concentrated HRP Conjugate:HRP Conjugate Diluent)

• Prepare the working solution immediately before use to maintain optimal enzyme activity

Storage Considerations:

• Store stock concentrated HRP conjugate according to manufacturer recommendations

• Do not store diluted working solutions for extended periods

• Avoid repeated freeze-thaw cycles of concentrated conjugate

Following these precise methodological steps ensures consistent enzymatic activity, minimizing inter-assay variability and maximizing detection sensitivity.

What are the critical factors for ensuring specificity in ANGPTL4 detection using antibody-based methods?

Ensuring high specificity in ANGPTL4 detection requires careful consideration of several technical factors:

Antibody Selection Criteria:

• Validation status: Select antibodies with validated specificity against ANGPTL4 in relevant species (human, mouse, rat)

• Domain specificity: Determine whether the antibody recognizes the N-terminal domain, C-terminal domain, or full-length protein based on experimental needs

• Cross-reactivity profile: Verify absence of significant cross-reactivity with related proteins

Experimental Controls to Ensure Specificity:

• Positive controls: Include known ANGPTL4-expressing tissues (placenta, hepatocellular carcinoma tissues)

• Negative controls: Include samples where primary antibody is omitted

• Competitive inhibition: Pre-incubation with recombinant ANGPTL4 should abolish signal

Sample Preparation Considerations:

• Optimal buffer systems to maintain ANGPTL4 conformational epitopes

• Appropriate blocking agents to minimize background

• Stringent washing steps to remove unbound antibodies

Implementing these methodological considerations significantly enhances the specificity and reliability of ANGPTL4 detection in research applications.

What is the optimal standard curve preparation for quantitative ANGPTL4 ELISA using HRP-based detection?

Generating a reliable standard curve is essential for accurate quantification of ANGPTL4 in biological samples. The following methodology ensures optimal standard curve preparation:

Standard Preparation Protocol:

• Reconstitute lyophilized ANGPTL4 standard according to manufacturer instructions

• Prepare a two-fold serial dilution series spanning 1.56-100 ng/mL concentration range

• Include a blank control (0 ng/mL) containing only diluent

Standard Curve Performance Characteristics:

Analytical Considerations:

• Sensitivity: The detection limit is typically 0.94 ng/mL

• Working range: The assay provides reliable quantification between 1.56-100 ng/mL

• Repeatability: Intra-assay coefficient of variation should be <10%

For accurate quantification, all standards and samples should be assayed in duplicate, and standard curves should be generated for each ELISA plate to account for plate-to-plate variations.

How should researchers interpret Western blot data showing multiple ANGPTL4 bands?

Interpreting Western blot data for ANGPTL4 requires careful consideration of post-translational modifications and proteolytic processing. Researchers commonly observe multiple bands on ANGPTL4 Western blots that require proper interpretation:

Common ANGPTL4 Western Blot Patterns:

• Full-length ANGPTL4: Often detected at ~65 kDa, despite theoretical molecular weight of 45.2 kDa due to glycosylation and other post-translational modifications

• N-terminal domain: ~26-28 kDa after proteolytic cleavage

• C-terminal domain: ~17-20 kDa after proteolytic cleavage

Interpretation Guidelines:

• Multiple bands may represent physiologically relevant processed forms rather than non-specific binding

• Post-translational modifications (particularly glycosylation) can cause significant shifts in apparent molecular weight

• Pro-protein convertase activity in the biological sample affects the ratio of full-length to cleaved forms

• Sample preparation conditions (reducing vs. non-reducing) can affect observed banding patterns

Methodological Approaches to Confirm Band Identity:

• Compare detection patterns using antibodies targeting different ANGPTL4 domains

• Include recombinant ANGPTL4 (full-length and domains) as positive controls

• Test lysates from tissues known to express different ANGPTL4 forms (placenta, liver, adipose tissue)

Understanding these considerations allows for accurate interpretation of ANGPTL4 Western blot data in experimental contexts.

How can researchers address discrepancies between ANGPTL4 ELISA and Western blot quantification?

Reconciling discrepancies between ELISA and Western blot quantification of ANGPTL4 requires understanding the fundamental differences between these techniques:

Common Sources of Discrepancies:

• Epitope Accessibility Differences:

  • ELISA typically detects native conformations

  • Western blot detects denatured epitopes

  • Some antibodies may preferentially recognize certain conformational states

• Domain-Specific Detection:

  • ELISA kits may be optimized to detect specific domains (N-terminal or C-terminal)

  • Western blot antibodies may have different domain specificity

  • The cleaved status of ANGPTL4 in different sample types affects detection

• Sample Matrix Effects:

  • ELISA may be subject to matrix interference from complex biological samples

  • Western blot involves SDS-PAGE separation that can reduce matrix effects

Methodological Approach to Reconcile Discrepancies:

• Determine domain specificity of both ELISA and Western blot antibodies

• Consider using recombinant standards containing both full-length and cleaved forms

• Validate results using orthogonal methods such as mass spectrometry

• When possible, use antibody pairs targeting the same epitopes for both methods

When significant discrepancies occur, researchers should report results from both methods with appropriate caveats about the limitations of each approach.

What are the critical considerations when analyzing ANGPTL4-LPL interactions in complex biological systems?

Analysis of ANGPTL4-lipoprotein lipase (LPL) interactions in complex biological systems requires consideration of several critical factors that influence experimental outcomes:

Physiological Variables Affecting ANGPTL4-LPL Interactions:

• Oligomerization State:

  • ANGPTL4 functions as oligomers, with different oligomeric forms exhibiting varying LPL inhibitory potency

  • The N-terminal domain mediates oligomerization and contains the LPL inhibitory activity

• Proteolytic Processing:

  • Cleavage of ANGPTL4 by pro-protein convertases enhances LPL inhibitory activity

  • The ratio of full-length to cleaved forms varies between tissues and physiological states

• Reversibility of Inhibition:

  • ANGPTL4 is a reversible, noncompetitive inhibitor of LPL

  • Inhibited LPL exists in complex with ANGPTL4 and can regain activity upon dissociation

Methodological Approaches for Studying These Interactions:

• In Vitro Systems:

  • Purified component assays to determine direct interactions

  • Kinetic studies to establish inhibition mechanisms

  • Structural analyses to identify interaction domains

• Cellular Systems:

  • Cell lines expressing defined ANGPTL4 and LPL variants

  • Analysis of triglyceride processing in presence of ANGPTL4

  • Co-immunoprecipitation to verify complex formation

• In Vivo Models:

  • Tissue-specific expression systems

  • Conditional knockout models

  • Physiological challenges (fasting, lipid loading) to assess dynamic regulation

Understanding these considerations enables researchers to design experiments that accurately reflect the complex regulatory mechanisms of lipid metabolism mediated by ANGPTL4-LPL interactions.

How can HRP-conjugated ANGPTL4 antibodies be utilized in multi-parameter imaging studies?

Advanced multi-parameter imaging approaches using HRP-conjugated ANGPTL4 antibodies enable simultaneous visualization of ANGPTL4 with other proteins or cellular structures. These methodologies provide valuable insights into protein co-localization and functional interactions.

Multiplexed Immunohistochemistry Protocols:

• Sequential Multiplex IHC:

  • Apply ANGPTL4 primary antibody and HRP-conjugated secondary

  • Develop with spectrally distinct chromogens (DAB for brown, AEC for red)

  • Strip or quench the first round of antibodies

  • Apply subsequent antibodies for other targets with different detection systems

• Tyramide Signal Amplification (TSA) Multiplexing:

  • Use HRP-conjugated antibodies with tyramide-fluorophore conjugates

  • HRP catalyzes covalent binding of fluorescent tyramides to nearby proteins

  • Heat-inactivate HRP after each round

  • Repeat with different antibodies and fluorophores

  • Enables detection of 5-7 proteins on a single tissue section

Methodological Considerations:

• Optimize antigen retrieval for all target proteins simultaneously

• Carefully validate antibody specificity in multiplex settings

• Include appropriate controls for signal bleeding and non-specific binding

• Use spectral unmixing for fluorescent applications to resolve overlapping signals

This approach has been successfully employed to visualize ANGPTL4 in relation to vascular structures and metabolic markers in various tissues, providing insights into its functional relationships in complex tissue environments.

What are the most promising analytical approaches for studying ANGPTL4 post-translational modifications?

Post-translational modifications (PTMs) of ANGPTL4 significantly impact its function and interactions. Advanced analytical approaches offer insights into these critical modifications:

Mass Spectrometry-Based Approaches:

• Bottom-up Proteomics:

  • Enzymatic digestion of ANGPTL4 followed by LC-MS/MS analysis

  • Identification of glycosylation, phosphorylation, and other modifications

  • Quantification of modification stoichiometry

• Top-down Proteomics:

  • Analysis of intact ANGPTL4 protein by high-resolution MS

  • Provides comprehensive view of proteoforms

  • Enables detection of combinatorial modifications

Site-Specific Analysis Techniques:

• Glycoprofiling:

  • Release and analysis of N-linked and O-linked glycans

  • Structural characterization using exoglycosidase digestions

  • Linking glycan structures to functional properties

• Phosphorylation Mapping:

  • Enrichment of phosphopeptides using titanium dioxide or IMAC

  • Identification of phosphorylation sites and their stoichiometry

  • Correlation with regulatory mechanisms

Integrated Analytical Workflow:

• Initial characterization using Western blot with modification-specific antibodies

• Confirmation and detailed mapping using MS-based approaches

• Functional studies using site-directed mutagenesis of modified residues

• Correlation of modification patterns with biological activity

These analytical approaches reveal how PTMs regulate ANGPTL4's ability to inhibit LPL and its interactions with other proteins, providing deeper insights into its role in lipid metabolism and other physiological processes.

What are the emerging trends in ANGPTL4 antibody applications for metabolic disease research?

Recent advances in ANGPTL4 antibody applications have opened new avenues for metabolic disease research. Several emerging trends demonstrate the expanding utility of these tools:

• Therapeutic Antibody Development:

  • Neutralizing antibodies targeting ANGPTL4 show promise for treating hypertriglyceridemia

  • Monoclonal antibodies specifically targeting the N-terminal domain can selectively modulate LPL inhibition

  • Combinatorial approaches targeting multiple angiopoietin-like proteins simultaneously

• Single-Cell Analysis Applications:

  • Integration of ANGPTL4 antibodies in mass cytometry (CyTOF) panels

  • Single-cell proteomics to examine ANGPTL4 expression heterogeneity

  • Spatial transcriptomics combined with antibody detection for tissue microenvironment analysis

• Methodological Innovations:

  • Development of proximity ligation assays to study ANGPTL4-protein interactions in situ

  • CRISPR-based screening with antibody readouts to identify ANGPTL4 regulators

  • Nanobody and aptamer alternatives to conventional antibodies for improved tissue penetration

These emerging applications are advancing our understanding of ANGPTL4's role in metabolic regulation and developing novel therapeutic strategies for metabolic disorders.

How can researchers integrate ANGPTL4 antibody data with multi-omics approaches?

Integration of ANGPTL4 antibody-based data with multi-omics approaches provides comprehensive insights into its regulatory networks and functional implications:

Multi-Omics Integration Strategies:

• Antibody-Based Proteomics with Transcriptomics:

  • Correlation of ANGPTL4 protein levels (detected by antibodies) with mRNA expression

  • Identification of post-transcriptional regulatory mechanisms

  • Discovery of discordant regulation between protein and transcript levels

• Functional Proteomics Integration:

  • Immunoprecipitation with ANGPTL4 antibodies followed by mass spectrometry

  • Identification of protein interaction networks

  • Correlation with functional metabolic outcomes

• Integrated Analysis Platforms:

  • Computational frameworks for multi-dimensional data integration

  • Network analysis of ANGPTL4 in lipid metabolism pathways

  • Systems biology approaches to model ANGPTL4 regulation

Methodological Workflow Example:

• Quantify ANGPTL4 using antibody-based methods (ELISA, IHC, WB)

• Perform RNA-seq or proteomics on the same samples

• Integrate with metabolomics data focusing on lipid profiles

• Apply computational methods to identify regulatory relationships