ANGPTL2 Antibody

What applications are most reliable for ANGPTL2 antibody-based detection?

Most commercial ANGPTL2 antibodies are validated for Western blotting (WB) and immunohistochemistry on paraffin-embedded sections (IHC-P). According to validation data, these applications show consistent results across different antibody products. Western blot analysis typically reveals ANGPTL2 at approximately 55-64 kDa under reducing conditions . For optimal Western blot results, concentrations between 0.5-2 μg/mL are recommended, though this should be optimized for your specific sample type .

For IHC-P applications, dilutions between 1:20 and 1:250 have been validated depending on the antibody . When performing immunohistochemistry with frozen sections, higher concentrations (5-15 μg/mL) may be necessary for optimal staining .

How should species reactivity be considered when selecting an ANGPTL2 antibody?

ANGPTL2 antibodies vary significantly in their species reactivity profiles:

When selecting an antibody, consider the sequence homology between species. For example, the immunogen for ABIN5518733 corresponds to amino acids 275-312 of human ANGPTL2, which differs from the mouse sequence by only one amino acid . This high conservation explains its cross-reactivity with multiple species. Always validate antibody performance in your specific species of interest, even when the manufacturer claims reactivity .

What are the critical differences between polyclonal and monoclonal ANGPTL2 antibodies?

Polyclonal ANGPTL2 antibodies:

• Most commercially available ANGPTL2 antibodies are polyclonal (typically rabbit or goat)

• Recognize multiple epitopes, potentially increasing detection sensitivity

• May show batch-to-batch variation that requires re-optimization

• Often generated using synthetic peptides or recombinant proteins as immunogens

Monoclonal ANGPTL2 antibodies:

• Available as mouse monoclonal antibodies (e.g., R&D Systems MAB2084)

• Provide consistent lot-to-lot reproducibility

• Recognize a single epitope, which may reduce background but potentially limit detection in some applications

• Often demonstrate greater specificity but potentially lower sensitivity than polyclonals

For studying specific domains of ANGPTL2, select antibodies generated against appropriate immunogens. For instance, antibodies targeting the middle region (aa 275-312) or N-terminal region have been commercialized for different research purposes .

How can I effectively use ANGPTL2 antibodies to investigate its dual role in tumor biology?

ANGPTL2's complex role in cancer progression requires careful experimental design. Horiguchi et al. demonstrated that ANGPTL2 can function as either a tumor promoter or suppressor depending on its cellular source .

Methodological approach:

• Cell-type specific analysis: Use co-immunostaining with ANGPTL2 antibodies and cell-type markers to determine which cells express ANGPTL2 in your tumor model. In B16-OVA melanoma models, stromal PDGFRα+ fibroblasts were identified as the primary source of ANGPTL2 using co-localization studies .

• Conditional knockout models: Employ cell-type specific knockout approaches to distinguish roles. For example, comparing renal tubular epithelial cell-specific versus systemic ANGPTL2 knockout in renal cell carcinoma models revealed opposing effects on tumor progression .

• Antibody selection for co-staining experiments: When investigating ANGPTL2 cellular sources, select antibodies compatible with multi-color immunofluorescence. For example, in the study by Horiguchi et al., they combined ANGPTL2 antibody staining with PDGFRα and PDGFRβ to identify the specific fibroblast population producing ANGPTL2 .

• Functional validation: After identifying ANGPTL2-producing cells, conduct functional studies by manipulating those specific cell populations to confirm their role in tumor progression or suppression .

What controls should be included when validating ANGPTL2 antibody specificity?

Proper controls are essential to ensure reliable ANGPTL2 detection:

• Positive tissue controls: Human heart, aorta, uterus tissues, and mouse intestinal tissue have been validated for ANGPTL2 expression . For cancer studies, human colon cancer tissue shows reliable ANGPTL2 expression .

• Negative controls:

  • Primary antibody omission

  • Isotype controls (rabbit IgG for polyclonal rabbit antibodies)

  • ANGPTL2 knockout or knockdown samples when available

• Cross-reactivity controls: Test against related proteins, particularly other angiopoietin-like family members. Good antibodies should show minimal cross-reactivity with ANGPTL1 and ANGPTL6 (<1%) .

• Peptide competition assays: Pre-incubate your antibody with the immunizing peptide before application to confirm specificity. The signal should be significantly reduced or eliminated.

• Multiple antibody validation: Compare staining patterns using antibodies raised against different ANGPTL2 epitopes to confirm consistent results .

How do I optimize immunohistochemical detection of ANGPTL2 in different tissue types?

Optimization strategies vary by tissue type and fixation method:

For paraffin-embedded tissues:

• Antigen retrieval: Most protocols require heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). Test both to determine optimal conditions for your tissue.

• Antibody concentration: Start with manufacturer recommendations (e.g., 1:20 dilution for ab199133 in human colon cancer tissue) , then optimize through titration experiments.

• Detection system optimization: For tissues with lower ANGPTL2 expression, amplification systems like tyramide signal amplification may enhance detection.

• Counterstain selection: When localizing ANGPTL2 in specific tissue compartments, select appropriate counterstains. In tumor microenvironment studies, using hematoxylin allows better visualization of tissue architecture .

For frozen sections:

• Higher antibody concentrations are typically required (5-15 μg/mL)

• Fixation method significantly impacts results – test 4% paraformaldehyde versus acetone fixation

• Block endogenous peroxidase activity before antibody incubation

Why might I observe multiple bands when using ANGPTL2 antibodies in Western blot?

Multiple bands in ANGPTL2 Western blots can occur for several biological and technical reasons:

• Post-translational modifications: ANGPTL2 is a glycoprotein with predicted molecular weight of 57 kDa, but glycosylation patterns can shift the apparent molecular weight to 55-64 kDa range .

• Proteolytic processing: ANGPTL2 may undergo proteolytic cleavage during sample preparation or as part of biological processing. Use protease inhibitors during sample preparation.

• Antibody specificity issues: Some antibodies may recognize related angiopoietin-like family members. Test specificity using recombinant proteins as controls – high-quality antibodies should show minimal cross-reactivity with ANGPTL1 or ANGPTL6 .

• Sample preparation artifacts: Incomplete denaturation or reduction can cause aggregation or incomplete protein separation. Ensure complete denaturation by heating samples at 95°C for 5 minutes in sample buffer containing SDS and reducing agents.

Validation data from R&D Systems shows ANGPTL2 detected at approximately 60 kDa in mouse uterus tissue and MEF cells using their AF1444 antibody , while human ANGPTL2 was detected at 55-64 kDa in heart, aorta, and uterus tissues using their AF2084 antibody .

How can I distinguish between ANGPTL2 expressed by tumor cells versus stromal cells?

Differentiating cellular sources of ANGPTL2 is critical for understanding its dual functions in cancer:

• Dual immunofluorescence staining:

  • Co-stain ANGPTL2 with cell-type specific markers:

  • Tumor cells: Specific tumor markers (e.g., cytokeratins for epithelial tumors)

  • Fibroblasts: PDGFRα, PDGFRβ, FAP, or ER-TR7

  • Immune cells: CD45 (leukocytes), CD68 (macrophages)

  • Endothelial cells: CD31

• Single-cell analysis approaches:

  • Laser capture microdissection followed by qRT-PCR for ANGPTL2

  • Single-cell RNA sequencing to identify cell populations expressing ANGPTL2

• In situ hybridization combined with immunohistochemistry:

  • RNAscope for ANGPTL2 mRNA combined with immunostaining for cell-type markers

In the study by Horiguchi et al., they demonstrated that in the B16-OVA melanoma model, ANGPTL2 was predominantly expressed by PDGFRα+ fibroblasts rather than tumor cells, PDGFRβ+ fibroblasts, CD45+ leukocytes, CD68+ macrophages, or CD31+ endothelial cells . This cellular source was critical for understanding its tumor-suppressive role in this context.

What experimental approaches can resolve contradictory findings about ANGPTL2 function?

Contradictions in ANGPTL2 research can be addressed through:

• Cell-type specific genetic manipulations:

  • Use conditional knockout models targeting specific cell types

  • For example, Horiguchi et al. compared renal tubular epithelial-specific ANGPTL2 knockout (using Chd16-Cre) versus systemic knockout, revealing opposing effects on tumor progression

• Temporal considerations:

  • Use inducible systems to control timing of ANGPTL2 manipulation

  • Early vs. late intervention may yield different results in cancer progression

• Comprehensive immune profiling:

  • Assess tumor-infiltrating immune cells by flow cytometry

  • Measure T-cell cross-priming and activation markers

  • Quantify tumor-specific CD8+ T cell responses using MHC tetramers

  • Analyze effector cytokine production (e.g., granzyme B)

• Mechanistic pathway analysis:

  • Investigate ANGPTL2 receptor engagement (integrin α5β1, PIR-B)

  • Examine downstream signaling pathways in different cell types

  • Test pathway inhibitors to confirm mechanistic hypotheses

Horiguchi et al. resolved contradictory findings by demonstrating that ANGPTL2 derived from PDGFRα+ fibroblasts enhanced anti-tumor immunity by promoting dendritic cell function and CD8+ T cell cross-priming, while tumor-derived ANGPTL2 promoted tumor progression through different mechanisms .

How do sample preparation methods affect ANGPTL2 detection?

Sample preparation critically impacts ANGPTL2 detection across methods:

For Western blot:

• Lysis buffers: RIPA buffer with protease inhibitors is commonly used for tissue lysates. For mouse intestinal tissue lysates, 40 μg total protein was sufficient for ANGPTL2 detection .

• Protein denaturation: Complete denaturation using 10% SDS-PAGE under reducing conditions is recommended . Incomplete denaturation may result in aggregation or aberrant migration patterns.

• Transfer conditions: PVDF membranes are preferred for ANGPTL2 detection . Standard wet transfer protocols (25V overnight at 4°C) typically yield optimal results.

For immunohistochemistry:

• Fixation impact: Formalin fixation may mask ANGPTL2 epitopes, requiring optimized antigen retrieval protocols. For frozen sections, brief fixation (10 minutes in 4% paraformaldehyde) generally preserves antigenicity.

• Optimal sectioning: For paraffin sections, 4-5 μm thickness is optimal; for frozen sections, 8-10 μm sections generally work well with ANGPTL2 antibodies.

• Blocking reagents: BSA-based blocking solutions (3-5%) are recommended to reduce background when using most ANGPTL2 antibodies.

What approaches can quantify ANGPTL2 levels in experimental and clinical samples?

Several quantitative approaches can be employed:

• Western blot quantification:

  • Densitometric analysis normalized to loading controls

  • Standard curves using recombinant ANGPTL2 protein

  • Typical detection range: 0.5-2 μg/mL antibody concentration for 40 μg total protein

• Immunohistochemical quantification:

  • H-score method (staining intensity × percentage of positive cells)

  • Digital image analysis using software like ImageJ or QuPath

  • Multiplex immunofluorescence for co-localization with other markers

• ELISA-based detection:

  • Commercial ELISA kits available for human and mouse ANGPTL2

  • Typical detection range: 0.2-20 ng/mL

  • Sample types: serum, plasma, cell culture supernatants

• RT-qPCR for mRNA expression:

  • For comparing expression levels between cell populations

  • Used by Horiguchi et al. to demonstrate that PDGFRα+ CAFs express higher ANGPTL2 mRNA levels than PDGFRα- CAFs

How can I investigate the relationship between ANGPTL2 and immune cell function in the tumor microenvironment?

Based on findings that stromal ANGPTL2 enhances anti-tumor immunity, several experimental approaches are recommended:

• Flow cytometric analysis of tumor-infiltrating lymphocytes:

  • Quantify CD8+ T cell infiltration

  • Measure activation markers (CD69, CD25)

  • Assess effector function (IFNγ, Granzyme B production)

• Antigen-specific T cell assays:

  • Use MHC tetramers to identify tumor-specific T cells (as demonstrated with OVA tetramers in the B16-OVA model)

  • Measure ex vivo responses to tumor antigens

  • Perform intracellular cytokine staining after peptide stimulation

• Dendritic cell function analysis:

  • Assess maturation markers (CD80, CD86, MHC II)

  • Examine cross-presentation capacity

  • Investigate PIR-B-NOTCH signaling pathway activation

• In vivo tumor vaccine models:

  • Compare vaccine efficacy in ANGPTL2-sufficient versus deficient hosts

  • Assess memory T cell formation and recall responses

  • Evaluate tumor re-challenge resistance

Horiguchi et al. demonstrated that ANGPTL2 deficiency in host stromal cells resulted in decreased numbers of tumor-infiltrating CD8+ T cells and reduced T-cell cross-priming in draining lymph nodes, supporting ANGPTL2's role in enhancing anti-tumor immunity .

How might ANGPTL2 antibodies be used to develop new cancer therapeutic strategies?

The dual role of ANGPTL2 suggests complex therapeutic applications:

• Targeted antibody approaches:

  • Cell-type specific delivery strategies to block tumor-derived ANGPTL2 while preserving stromal ANGPTL2

  • Domain-specific antibodies that selectively inhibit pro-tumorigenic functions while preserving immunostimulatory effects

• Combination with immunotherapy:

  • ANGPTL2 supplementation may enhance dendritic cell function and CD8+ T cell priming

  • Potential synergy with checkpoint inhibitors by promoting T cell infiltration and activation

  • Therapeutic vaccines could be enhanced by ANGPTL2 co-administration

• Biomarker development:

  • Monitoring circulating ANGPTL2 levels during treatment

  • Tissue-based assessment of ANGPTL2 source (tumor vs. stromal) to guide treatment decisions

  • Ratio of tumor to stromal ANGPTL2 as a potential prognostic indicator

• Cell-based therapies:

  • Engineering dendritic cells to respond optimally to ANGPTL2 signaling

  • Modifying CAR-T cells to overcome ANGPTL2-mediated immunosuppression in certain contexts

The discovery that ANGPTL2 activates dendritic cells through PIR-B–NOTCH signaling and enhances tumor vaccine efficacy suggests potential applications in cancer vaccine development .

What are the most promising research models for studying ANGPTL2 function in disease?

Based on current research, several models show particular promise:

• Syngeneic mouse tumor models:

  • B16-OVA melanoma model allows clear distinction between host and tumor-derived ANGPTL2

  • Permits detailed study of immune responses using defined tumor antigens

• Genetic cancer models:

  • PRCC-TFE3 translocation renal cell carcinoma model demonstrated upregulation of ANGPTL2 and revealed its dual functions

  • Conditional knockout approaches using tissue-specific Cre drivers provide mechanistic insights

• Patient-derived xenografts:

  • Can maintain tumor heterogeneity and stromal components

  • Allow testing of human-specific ANGPTL2 antibodies

  • Useful for translational studies bridging mouse findings to human disease

• In vitro co-culture systems:

  • Tumor cells with fibroblasts, immune cells, and endothelial cells

  • Allow mechanistic dissection of ANGPTL2 signaling between cell types

  • Useful for high-throughput screening of therapeutic approaches

• 3D organoid cultures:

  • Preserve tissue architecture and cellular heterogeneity

  • Enable studies of ANGPTL2 in specific tissue microenvironments

  • Allow genetic manipulation in physiologically relevant systems