ANGPTL3 (PAT23D8AT) Antibody

What is the ANGPTL3 (PAT23D8AT) Antibody and what are its key specifications?

The ANGPTL3 (PAT23D8AT) Antibody (CPAB0087) is a mouse monoclonal antibody developed for research applications. It is derived from hybridization of mouse F myeloma cells with spleen cells from BALB/c mice immunized with recombinant human ANGPTL3 protein (amino acids 243-46) . The antibody has the following technical specifications:

The antibody targets human ANGPTL3 with high specificity, making it suitable for various research applications investigating lipid metabolism and cardiovascular biology .

What is the biological significance of ANGPTL3 in lipid metabolism research?

ANGPTL3 (angiopoietin-like protein 3) is a liver-secreted protein that plays a critical role in regulating triglyceride metabolism by inhibiting lipoprotein lipase (LPL), the enzyme responsible for hydrolyzing triglycerides in plasma lipoproteins . Research has established several key aspects of ANGPTL3 biology:

• It regulates circulating triglyceride levels during different nutritional states (feeding/fasting) through differential inhibition of LPL .

• ANGPTL3 works in conjunction with ANGPTL8, forming a complex that has enhanced inhibitory effects on LPL activity compared to ANGPTL3 alone .

• Recent research indicates ANGPTL3 preferentially resides on high-density lipoprotein (HDL) particles, affecting its functional properties .

• Plasma ANGPTL3 concentrations show positive association with coronary artery disease, suggesting its role as a biomarker or therapeutic target .

• The N-terminal domain (fragment 17-207) increases plasma triglyceride levels, while the C-terminal fibrinogen-like domain does not appear to have this effect .

These characteristics make ANGPTL3 an important target for research in dyslipidemia, atherosclerosis, and cardiovascular disease.

How does the ANGPTL3/ANGPTL8 complex differ functionally from ANGPTL3 alone?

The ANGPTL3/ANGPTL8 complex exhibits distinct functional properties compared to ANGPTL3 alone, with important implications for research design:

• The complex demonstrates enhanced LPL inhibitory activity compared to either protein individually, with optimal activity observed at a ratio of 3 ANGPTL3 molecules per 1 ANGPTL8 molecule .

• Research using hydrogen-deuterium exchange mass spectrometry (HDXMS) and molecular modeling has identified a specific leucine zipper-like motif within the ANGPTL3/8 complex that serves as the LPL-inhibitory region .

• This region also appears to be the ApoA5-interacting region, suggesting competitive binding between ApoA5 and LPL for the same ANGPTL3/8 binding site .

• Anti-ANGPTL3/8 antibodies specifically targeting the complex (rather than individual proteins) have been shown to potently block ANGPTL3/8-mediated LPL inhibition in vitro and significantly lower triglyceride levels in vivo .

Understanding these differences is crucial when designing experiments with the PAT23D8AT antibody, as its effects may differ depending on whether it recognizes free ANGPTL3 or the ANGPTL3/8 complex.

What are the recommended protocols for using ANGPTL3 (PAT23D8AT) Antibody in Western Blot applications?

For optimal Western Blot results with the ANGPTL3 (PAT23D8AT) Antibody, follow these methodological steps:

• Sample preparation: Prepare cell or tissue lysates in a compatible lysis buffer containing protease inhibitors (critical for preserving ANGPTL3 integrity).

• Protein quantification: Determine protein concentration using Bradford or BCA assay.

• SDS-PAGE: Separate proteins using 10-12% polyacrylamide gels (ANGPTL3 has a molecular weight of approximately 60-70 kDa).

• Transfer: Transfer proteins to a PVDF or nitrocellulose membrane using standard protocols.

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

• Primary antibody incubation: Dilute ANGPTL3 (PAT23D8AT) Antibody (starting dilution 1:1000) in blocking buffer and incubate overnight at 4°C.

• Washing: Wash membrane 3-5 times with TBST, 5 minutes per wash.

• Secondary antibody: Incubate with HRP-conjugated anti-mouse IgG (typically 1:5000) for 1 hour at room temperature.

• Detection: Apply ECL substrate and visualize using film or digital imaging systems.

Always include positive controls (such as HepG2 cell lysates or human liver tissue samples) that express ANGPTL3 and optimize antibody concentration for your specific experimental conditions .

How should researchers optimize ELISA protocols with the ANGPTL3 (PAT23D8AT) Antibody?

When developing ELISA protocols using the ANGPTL3 (PAT23D8AT) Antibody, consider these methodological approaches:

• For sandwich ELISA:

  • Coat plates with a capture antibody specific to a different ANGPTL3 epitope

  • Use PAT23D8AT as detection antibody (1:2000 dilution recommended initially)

  • Include a standard curve using recombinant human ANGPTL3 (243-460 amino acids)

• For direct ELISA:

  • Coat plates with diluted samples containing ANGPTL3

  • Detect bound ANGPTL3 using PAT23D8AT antibody

  • Follow with appropriate secondary antibody and detection system

• For competitive ELISA:

  • Pre-incubate samples with PAT23D8AT antibody

  • Add to plates coated with recombinant ANGPTL3

  • Detect unbound antibody with secondary antibody

The human ANGPTL3 DuoSet ELISA system (as used in research studies) provides a reliable framework that can be adapted for use with the PAT23D8AT antibody . When analyzing plasma samples, be aware that ANGPTL3 associates with lipoproteins, which may affect detection efficiency in different assay formats .

What are the key considerations for studying ANGPTL3-lipoprotein interactions using the PAT23D8AT antibody?

Recent research has revealed ANGPTL3 preferentially resides on high-density lipoprotein (HDL) particles, which has important implications for experimental design . When studying these interactions:

• Lipoprotein fractionation approaches:

  • Use FPLC (Fast Protein Liquid Chromatography) to separate plasma lipoprotein fractions

  • Apply PAT23D8AT antibody in Western blot analysis of fractions to detect ANGPTL3 distribution

  • Consider that ANGPTL3 is approximately 4-fold enriched on LDL in certain conditions, as observed in subjects with ABCA1 deficiency

• Functional impact assessment:

  • Studies show unbound ANGPTL3 does not suppress lipase activity, while ANGPTL3 combined with HDL or LDL suppresses activity by 21.4% and 25.4%, respectively

  • Design experiments using T37i brown adipocytes (which express LPL) to measure 3H-trioleate hydrolysis in the presence of ANGPTL3 with or without lipoproteins

  • Compare the antibody's ability to neutralize ANGPTL3 function in different lipoprotein contexts

• Technical considerations:

  • Optimize sample handling to preserve native lipoprotein-ANGPTL3 associations

  • Consider using native rather than denaturing conditions for certain applications

  • Validate whether PAT23D8AT recognizes lipoprotein-bound ANGPTL3 with the same efficiency as free ANGPTL3

These approaches will help elucidate the physiological relevance of ANGPTL3-lipoprotein interactions and their role in lipid metabolism regulation.

How can the PAT23D8AT antibody be utilized to evaluate ANGPTL3 inhibition strategies?

The PAT23D8AT antibody can serve as a valuable tool for evaluating ANGPTL3 inhibition strategies through several advanced applications:

• Comparing inhibition mechanisms:

  • Use PAT23D8AT to detect ANGPTL3 levels following treatment with various inhibitors (other antibodies, small molecules, genetic approaches)

  • Apply the antibody in Western blot or ELISA to quantify ANGPTL3 reduction in plasma or tissue samples

  • Determine whether inhibitors affect ANGPTL3 production, secretion, or clearance

• Epitope mapping and functional domain analysis:

  • Determine if PAT23D8AT binds to functionally relevant domains of ANGPTL3

  • Compare with established therapeutic antibodies like REGN1500, which binds ANGPTL3 from multiple species with high affinity (KD = 0.26–1.28 nM)

  • Use competitive binding assays to map the PAT23D8AT epitope relative to the LPL-binding region

• In vitro inhibition validation:

  • Assess whether PAT23D8AT itself can block ANGPTL3-mediated inhibition of LPL

  • Use recombinant LPL activity assays to quantify any inhibitory potency

  • Compare with known ANGPTL3 inhibitors like REGN1500, which has been shown to reverse ANGPTL3-induced inhibition of LPL activity in vitro and reduce plasma TG levels by ≥50% in vivo

• Biomarker development:

  • Utilize PAT23D8AT in ELISA assays to measure circulating ANGPTL3 levels

  • Correlate ANGPTL3 levels with lipid parameters and cardiovascular outcomes

  • Track changes in ANGPTL3 concentration during therapeutic interventions

Understanding how different inhibitory approaches affect ANGPTL3 function provides valuable insights for developing therapeutic strategies targeting lipid metabolism disorders.

What approaches can researchers use to study the interaction between ANGPTL3 and ANGPTL8 using the PAT23D8AT antibody?

The interaction between ANGPTL3 and ANGPTL8 is crucial for regulating LPL activity. To study this interaction using the PAT23D8AT antibody:

• Complex formation analysis:

  • Prepare ANGPTL3/8 complex in vitro using recombinant proteins (optimal ratio: 3 ANGPTL3 molecules per 1 ANGPTL8 molecule)

  • Use PAT23D8AT in immunoprecipitation experiments to pull down ANGPTL3 and detect co-precipitated ANGPTL8

  • Assess whether PAT23D8AT binding affects complex formation or stability

• Epitope accessibility studies:

  • Determine if the PAT23D8AT epitope remains accessible in the ANGPTL3/8 complex

  • Compare binding affinity to free ANGPTL3 versus the ANGPTL3/8 complex using bio-layer interferometry

  • Similar to approaches used with therapeutic antibodies, immobilize PAT23D8AT on streptavidin biosensors and measure binding to ANGPTL3, ANGPTL8, or ANGPTL3/8 complex (5 μg/ml)

• Functional impact assessment:

  • Evaluate whether PAT23D8AT affects the enhanced LPL inhibitory activity of the ANGPTL3/8 complex

  • Compare with antibodies specifically targeting the complex rather than individual proteins

  • Test in LPL activity assays similar to those used for anti-ANGPTL3/8 antibodies that have shown potent blocking of ANGPTL3/8-mediated LPL inhibition in vitro

• Cellular studies:

  • Assess PAT23D8AT effects in systems expressing both ANGPTL3 and ANGPTL8

  • Consider using T37i brown adipocytes that express LPL for functional studies

  • Measure changes in LPL activity and triglyceride hydrolysis

These approaches will provide insights into the molecular mechanisms of ANGPTL3/8 complex formation and function.

How can bio-layer interferometry be adapted for investigating ANGPTL3 antibody interactions?

Bio-layer interferometry (BLI) represents a powerful approach for studying interactions between ANGPTL3, its binding partners, and antibodies. Based on published methodologies:

• Basic binding characterization:

  • Immobilize PAT23D8AT antibody on streptavidin biosensors

  • Measure binding to ANGPTL3, ANGPTL8, or ANGPTL3/8 complex (5 μg/ml each)

  • Transfer to buffer-only wells to monitor dissociation kinetics

  • Calculate association and dissociation rate constants (ka and kd) and equilibrium dissociation constant (KD)

• Complex interaction studies:

  • Immobilize avidin-tagged LPL copurified with GPIHBP1 on streptavidin biosensors

  • Study the interaction with ANGPTL3/8 in the absence or presence of PAT23D8AT

  • Determine if the antibody blocks ANGPTL3-LPL interactions

• Epitope mapping:

  • Use reference antibodies with known binding sites as controls

  • Perform sequential binding experiments to identify overlapping epitopes

  • Compare binding profiles with therapeutic antibodies like REGN1500 that have KD values of 0.26–1.28 nM for ANGPTL3 from various species

• Quantitative analysis:

  • Process data using evaluation software (e.g., Octet RED96e® software)

  • Calculate kinetic parameters using models that account for complex binding interactions

  • Compare affinity and kinetics across different experimental conditions

This approach provides quantitative insights into antibody-antigen interactions with high precision and reproducibility, allowing researchers to characterize PAT23D8AT binding properties in detail.

What are common challenges in detecting ANGPTL3 in biological samples and how can they be addressed?

Researchers may encounter several challenges when detecting ANGPTL3 in biological samples:

• Lipoprotein association effects:

  • ANGPTL3 preferentially associates with HDL particles, which may mask epitopes or affect antibody binding

  • Solution: Consider using detergents that preserve epitope accessibility while releasing ANGPTL3 from lipoproteins

  • Alternative approach: Fractionate samples prior to analysis to separate free and lipoprotein-bound ANGPTL3

• Variable expression levels:

  • ANGPTL3 expression shows considerable variation between tissues and under different physiological conditions

  • Solution: Adjust sample concentration and optimize detection sensitivity

  • Consider using liver-derived cell lines like HepG2 as positive controls, as the liver is the primary source of ANGPTL3

• Complex formation with ANGPTL8:

  • ANGPTL3/8 complex formation may alter epitope accessibility

  • Solution: Use denaturing conditions in Western blot applications if detecting total ANGPTL3 is the goal

  • For studying the complex specifically, use native conditions and optimize antibody concentrations

• Post-translational modifications:

  • Glycosylation may affect antibody recognition

  • Solution: Consider enzymatic deglycosylation treatments if inconsistent results are observed

  • Compare results with antibodies targeting different epitopes

• Sample preparation artifacts:

  • Proteolytic degradation during sample processing may yield inconsistent results

  • Solution: Include protease inhibitors in all extraction buffers

  • Process samples consistently and avoid repeated freeze-thaw cycles

Addressing these challenges will improve the reliability and consistency of ANGPTL3 detection in research applications.

How should researchers interpret differences between ANGPTL3 inhibition by PAT23D8AT and other ANGPTL3-targeting approaches?

When comparing results from different ANGPTL3 inhibition approaches, researchers should consider:

• Mechanism of action differences:

  • PAT23D8AT may neutralize ANGPTL3 without affecting its plasma levels

  • Genetic approaches (siRNA, CRISPR/Cas9) reduce ANGPTL3 production

  • Therapeutic antibodies like REGN1500 bind ANGPTL3 with high affinity (KD = 0.26–1.28 nM) and neutralize its function

  • These mechanism differences may lead to variable effects on different ANGPTL3 functions

• Epitope-specific effects:

  • Different antibodies target distinct epitopes that may have variable importance for different ANGPTL3 functions

  • PAT23D8AT may affect ANGPTL3-LPL interaction differently than ANGPTL3-EL interaction

  • REGN1500, for example, affects both LPL and EL inhibition by ANGPTL3

• Temporal considerations:

  • Antibody-mediated inhibition provides acute, reversible effects

  • Genetic approaches produce sustained inhibition

  • Different approaches may yield different results depending on measurement timing

• Dosage effects:

  • REGN1500 reduces plasma TG by ≥50% in mice , providing a benchmark for comparison

  • Dose-response relationships may differ between inhibitors

  • Consider titrating PAT23D8AT concentration to establish comparable inhibition levels

• Context-dependent outcomes:

  • Effects may differ between normolipidemic and dyslipidemic models

  • REGN1500 reduces TG, LDL-C, and HDL-C in dyslipidemic mice without affecting tissue TG content

  • PAT23D8AT effects may similarly depend on baseline metabolic state

Understanding these factors allows for more accurate interpretation of experimental results and better comparison across different inhibition strategies.

What controls should be included when using ANGPTL3 (PAT23D8AT) Antibody in research applications?

To ensure experimental rigor when using the ANGPTL3 (PAT23D8AT) Antibody, include these essential controls:

• For Western blot applications:

  • Positive control: HepG2 cell lysates or human liver tissue (primary ANGPTL3 expression site)

  • Negative control: Tissues known not to express ANGPTL3 or ANGPTL3-knockout samples

  • Loading control: Housekeeping protein (β-actin, GAPDH) to normalize expression

  • Isotype control: Mouse IgG2a at the same concentration as PAT23D8AT

  • Blocking peptide: Pre-incubate antibody with recombinant ANGPTL3 to confirm specificity

• For ELISA applications:

  • Standard curve: Serial dilutions of recombinant human ANGPTL3

  • Blank control: All reagents except primary antibody

  • Sample dilution validation: Test several dilutions to ensure linearity

  • Spike recovery test: Add known amounts of recombinant ANGPTL3 to samples

  • Cross-reactivity test: Ensure no signal with related proteins (ANGPTL4, ANGPTL8)

• For functional inhibition studies:

  • ANGPTL3 alone control: Establish baseline ANGPTL3 activity

  • ANGPTL3/8 complex: Compare effects on complex vs. ANGPTL3 alone

  • Lipoprotein controls: Test with and without HDL/LDL, as they affect ANGPTL3 activity

  • Positive inhibition control: Include known ANGPTL3 inhibitor like REGN1500

  • Dose-response analysis: Test multiple antibody concentrations

• For specificity validation:

  • Test reactivity against recombinant ANGPTL3 from different species

  • Confirm lack of binding to other ANGPTL family members

  • Validate with orthogonal detection methods (e.g., mass spectrometry)

These controls will help ensure reliable, reproducible, and scientifically rigorous results when working with the ANGPTL3 (PAT23D8AT) Antibody.

How might ANGPTL3 (PAT23D8AT) Antibody be used to investigate novel aspects of ANGPTL3 biology?

The ANGPTL3 (PAT23D8AT) Antibody offers opportunities to explore several emerging areas of ANGPTL3 biology:

• HDL functionality studies:

  • Recent findings show ANGPTL3 preferentially resides on HDL particles

  • PAT23D8AT could be used to study how ANGPTL3 affects HDL composition and function

  • Investigate whether ANGPTL3-enriched HDL has altered cholesterol efflux capacity or anti-inflammatory properties

• Tissue-specific ANGPTL3 functions:

  • Beyond circulating ANGPTL3, investigate local production and actions in tissues

  • Use immunohistochemistry with PAT23D8AT to map tissue distribution

  • Correlate local ANGPTL3 levels with tissue-specific lipid metabolism markers

• Interaction with ANGPTL8 regulation:

  • The ANGPTL3/8 complex has enhanced LPL inhibitory activity

  • Investigate how nutritional, hormonal, or pathological conditions affect complex formation

  • Use PAT23D8AT to immunoprecipitate ANGPTL3 and assess ANGPTL8 co-precipitation under various conditions

• Metabolic disease mechanisms:

  • ANGPTL3 plasma levels positively associate with coronary artery disease (odds ratio, 1.25 [95% CI, 1.01–1.55])

  • Investigate ANGPTL3 as a biomarker for cardiovascular risk stratification

  • Study how interventions that alter ANGPTL3 levels affect disease progression

• Lipoprotein interaction mechanisms:

  • Explore structural requirements for ANGPTL3 binding to lipoproteins

  • Investigate whether PAT23D8AT affects ANGPTL3-lipoprotein associations

  • Study competitive interactions between lipoproteins for ANGPTL3 binding

These research directions would advance our understanding of ANGPTL3 biology beyond its established role in lipid metabolism.

How can the PAT23D8AT antibody complement genetic approaches to studying ANGPTL3 function?

The PAT23D8AT antibody can synergize with genetic approaches to provide comprehensive insights into ANGPTL3 function:

• Temporal control advantages:

  • Genetic knockouts provide complete loss of function from development

  • PAT23D8AT can be applied at specific time points to study acute ANGPTL3 neutralization

  • This combination helps distinguish developmental versus acute roles of ANGPTL3

• Partial versus complete inhibition:

  • Genetic approaches typically eliminate ANGPTL3 completely

  • Antibody-mediated neutralization can be titrated for partial inhibition

  • This allows dose-response studies impossible with genetic knockouts alone

• Structure-function analysis:

  • Generate ANGPTL3 mutants with modified domains or binding sites

  • Use PAT23D8AT to determine if mutations affect antibody binding

  • Correlate binding changes with functional alterations to map critical domains

• Compensation mechanism identification:

  • Genetic knockouts may trigger compensatory mechanisms over time

  • Acute antibody application helps identify immediate effects before compensation occurs

  • Compare acute antibody effects in wild-type versus heterozygous ANGPTL3+/- models

• Translational relevance:

  • Therapeutic approaches target existing ANGPTL3 rather than preventing expression

  • Antibody studies better mimic potential therapeutic interventions

  • Combine with genetic approaches to predict responders versus non-responders to therapy

This complementary approach yields more comprehensive understanding than either method alone and has greater translational relevance to human therapeutic development.