SFRP2 Antibody, HRP conjugated

What is SFRP2 and why is it a significant target for antibody-based research?

SFRP2 (Secreted frizzled-related protein 2) functions as a critical modulator of Wnt signaling through direct interaction with Wnt proteins, regulating cell growth and differentiation in specific cell types. It is widely expressed in various tissues including the heart, lungs, and kidneys, and plays important roles in eye retinal development and myogenesis . SFRP2 has gained significant research interest because it is overexpressed in the vasculature of approximately 85% of human breast tumors and other cancer types . Its involvement in promoting angiogenesis, tumor cell migration, and immunomodulation makes it an important target for both basic research and therapeutic development .

What are the key applications for SFRP2 antibodies in laboratory research?

SFRP2 antibodies are valuable tools for multiple research applications:

• Western Blotting (WB): For detecting endogenous levels of SFRP2 protein in tissue or cell lysates

• Immunohistochemistry (IHC): For visualizing SFRP2 expression patterns in tissue sections

• ELISA: For quantifying SFRP2 levels in biological fluids or cell culture media

• Dot Blot Analysis: For rapid screening of SFRP2 in samples

• Immunofluorescence (IF): For subcellular localization studies of SFRP2

HRP-conjugated SFRP2 antibodies specifically enhance sensitivity in WB, ELISA, and IHC applications by eliminating the need for secondary antibody incubation, reducing background, and enabling direct detection through enzymatic color development or chemiluminescence.

What are the recommended protocols for optimizing SFRP2 antibody, HRP conjugated in Western blotting?

For optimal Western blotting results with HRP-conjugated SFRP2 antibodies:

• Sample Preparation:

  • Use RIPA buffer with protease inhibitors for tissue/cell lysis

  • Load 20-50 μg of total protein per lane

• Optimization Steps:

  • Begin with a 1:1000 dilution (approximately 0.5 μg/ml) in 5% BSA/TBST

  • Incubate membrane overnight at 4°C with gentle agitation

  • Perform stringent washing (4-5 times with TBST)

  • Use enhanced chemiluminescence detection with varying exposure times

• Controls:

  • Include recombinant human SFRP2 protein as a positive control

  • Test for cross-reactivity with other SFRP family members (particularly SFRP1)

Note that antibody specificity testing has shown that some SFRP2 antibodies do not cross-react with human SFRP1, making them ideal for discriminating between these related proteins .

How can SFRP2 antibodies be used to investigate SFRP2's role in cancer progression models?

SFRP2 antibodies are critical tools for elucidating SFRP2's role in cancer progression through multiple experimental approaches:

• Tumor Microenvironment Analysis:

  • Use SFRP2 antibodies (HRP-conjugated) for immunohistochemical analysis of tumor sections to identify SFRP2 distribution in cancer tissues and correlate with vasculature markers (CD31, CD34)

  • Double staining procedures can reveal colocalization with endothelial markers

• Mechanistic Studies:

  • Apply SFRP2 antibodies to investigate signaling pathways in tumor and endothelial cells

  • Monitor effects on β-catenin and NFATc3 activation, as SFRP2 antagonism results in inhibition of both pathways

• In Vivo Models:

  • SFRP2 monoclonal antibody treatment of SVR angiosarcoma allografts in nude mice has been shown to decrease tumor volume by 58% compared with control (P = 0.004)

  • Similar treatment of MDA-MB-231 breast carcinoma xenografts decreased tumor volume by 52% (P = 0.03)

These applications highlight the dual utility of SFRP2 antibodies as both research tools and potential therapeutic agents.

What methodological approaches should be considered when using SFRP2 antibodies to study its impact on angiogenesis?

When investigating SFRP2's role in angiogenesis, researchers should consider these methodological approaches:

• In Vitro Assays:

  • Endothelial cell tube formation assays: Plate endothelial cells on Matrigel, treat with SFRP2 protein or anti-SFRP2 antibodies, and quantify tube formation after 4-16 hours

  • Endothelial cell migration assays: Use scratch wound or transwell migration assays to assess SFRP2's effects on endothelial motility

  • Apoptosis assays: Evaluate SFRP2's protective effects against hypoxia-induced endothelial apoptosis

• In Vivo Models:

  • Mouse Matrigel plug assay: Mix SFRP2 protein or antibodies with Matrigel and inject subcutaneously; after 7-14 days, harvest plugs for histological analysis of vessel formation

  • Chick chorioallantoic membrane (CAM) assay: Apply SFRP2 protein or antibodies to the CAM and quantify vessel formation

• Molecular Pathway Analysis:

  • Use HRP-conjugated SFRP2 antibodies in Western blotting to detect changes in NFATc3 activation, as SFRP2 stimulates angiogenesis via the calcineurin/NFAT signaling pathway

These methodologies provide complementary approaches to comprehensively evaluate SFRP2's angiogenic functions and the efficacy of anti-SFRP2 strategies.

How can SFRP2 antibodies be utilized to study immune responses in the tumor microenvironment?

Recent research has revealed SFRP2's unexpected roles in immune regulation, making SFRP2 antibodies valuable tools for studying tumor immunology:

• T-cell Function Analysis:

  • Flow cytometry with SFRP2 antibodies can assess SFRP2 expression on T-cells and correlate with T-cell exhaustion markers

  • Co-culture experiments with tumor cells and T-cells can evaluate how SFRP2 blockade affects T-cell proliferation and function

• Immune Checkpoint Regulation:

  • SFRP2 promotes NFATc3, CD38, and PD-1 expression in T-cells

  • SFRP2 antibody treatment counteracts these effects and increases NAD+ levels

  • Western blot analysis using HRP-conjugated antibodies can track changes in these markers following intervention

• Combined Immunotherapy Approaches:

  • Studies show SFRP2 blockade can overcome resistance to PD-1 inhibitors

  • When PD-1 mAb alone had minimal effect, hSFRP2 mAb combination with PD-1 mAb demonstrated an additive antimetastatic effect

These applications highlight SFRP2 antibodies' value in understanding the intersection between angiogenesis and immunomodulation in the tumor microenvironment.

What methodological considerations are important when using SFRP2 antibodies to evaluate treatment efficacy in tumor models?

When evaluating anti-SFRP2 treatment efficacy, researchers should consider these methodological approaches:

• Serum Biomarker Analysis:

  • Use ELISA with HRP-conjugated SFRP2 antibodies to monitor SFRP2 levels in serum before and after treatment

  • Treatment efficacy correlates with lower SFRP2 levels in serum, reduced CD38 levels in tumor-infiltrating lymphocytes, and lower PD-1 levels in T-cells

• Tumor Response Assessment:

  • Measure tumor volume longitudinally using calipers or advanced imaging

  • Perform immunohistochemistry on tumor sections to assess:

    • Microvessel density (CD31 staining)

    • Tumor cell apoptosis (TUNEL or cleaved caspase-3)

    • Immune cell infiltration (CD3, CD8, etc.)

• Pharmacokinetic/Biodistribution Analysis:

  • Studies show anti-SFRP2 antibodies are long-circulating in the blood and preferentially accumulate in SFRP2-positive tumors

  • Use labeled antibodies to track tissue distribution and tumor penetration

These methodologies provide multiple complementary endpoints to thoroughly evaluate SFRP2-targeted therapies.

What are the critical quality control parameters for validating SFRP2 antibody, HRP conjugated before experimental use?

Proper antibody validation ensures reliable experimental results. For HRP-conjugated SFRP2 antibodies, researchers should verify:

• Specificity Testing:

  • Perform dot blot analysis against recombinant human SFRP2 protein

  • Test cross-reactivity with related proteins, particularly other SFRP family members

  • Ideal antibodies should detect SFRP2 without cross-reacting with SFRP1

• Sensitivity Assessment:

  • Serial dilution testing to determine the limit of detection

  • Signal-to-noise ratio evaluation across different applications

• HRP Conjugation Quality:

  • Verify enzymatic activity through chemiluminescent substrate testing

  • Check protein-to-HRP molar ratio (typically 1:1 to 1:4)

  • Test storage stability at various temperatures (4°C, -20°C, -80°C)

• Application-Specific Controls:

  • For Western blotting: Positive control (recombinant human SFRP2) and negative control tissues

  • For IHC: Known positive tissue sections and appropriate blocking controls

These validation steps ensure reproducible results and minimize artifacts in subsequent experiments.

How can researchers troubleshoot common issues with HRP-conjugated SFRP2 antibodies in immunohistochemistry applications?

Common challenges with HRP-conjugated SFRP2 antibodies in IHC and their solutions include:

• High Background Signal:

  • Increase blocking duration (use 5-10% normal serum from the same species as the secondary antibody)

  • Add 0.1-0.3% Triton X-100 to blocking solution to reduce non-specific binding

  • Optimize antibody concentration through titration experiments

  • Ensure complete quenching of endogenous peroxidase activity (use 0.3% H₂O₂ in methanol for 30 minutes)

• Weak or Absent Staining:

  • Optimize antigen retrieval (test multiple methods: heat-induced with citrate buffer pH 6.0 or EDTA buffer pH 8.0)

  • Increase antibody concentration or incubation time

  • Ensure tissue fixation protocols are optimized (overfixation can mask epitopes)

  • Try signal amplification systems (e.g., avidin-biotin complex)

• Non-uniform Staining:

  • Ensure adequate tissue section thickness (4-6 μm ideal)

  • Check for complete deparaffinization and rehydration

  • Apply antibody solution to completely cover the tissue

  • Use humidity chambers during incubation

• Degradation of HRP Activity:

  • Store antibody according to manufacturer recommendations (typically 4°C for short-term, -20°C with glycerol for long-term)

  • Avoid repeated freeze-thaw cycles

  • Use freshly prepared DAB substrate solution

These troubleshooting approaches help maximize the utility of HRP-conjugated SFRP2 antibodies in tissue-based applications.

How can SFRP2 antibodies contribute to understanding the interplay between Wnt signaling and other pathways in disease models?

SFRP2 antibodies are valuable tools for dissecting complex signaling relationships:

• Canonical vs. Non-canonical Wnt Pathway Analysis:

  • SFRP2 has conflicting reports as either an antagonist or agonist of β-catenin

  • Use HRP-conjugated SFRP2 antibodies to track protein interactions and pathway activation

  • In endothelial cells, the canonical Wnt pathway is not affected by SFRP2, but non-canonical Wnt/Calcium signaling is activated

• NFATc3 Pathway Investigation:

  • SFRP2 antagonism inhibits NFATc3 activation in both endothelial and tumor cells

  • HRP-conjugated SFRP2 antibodies can be used in Western blots to monitor NFATc3 nuclear translocation and activation

• Multi-pathway Analysis:

  • Combination of SFRP2 antibodies with other pathway-specific antibodies in multiplexed assays

  • Proteomic approaches using SFRP2 antibodies for immunoprecipitation followed by mass spectrometry

These approaches help unravel the complex signaling network around SFRP2 and identify potential therapeutic vulnerabilities.

What are the methodological approaches for using SFRP2 antibodies to study its role in therapeutic resistance mechanisms?

Research indicates SFRP2's involvement in therapeutic resistance, particularly to immunotherapy. Key methodological approaches include:

• Resistance Model Development:

  • Create PD-1 inhibitor-resistant cell lines or animal models

  • Use HRP-conjugated SFRP2 antibodies to monitor SFRP2 expression changes during resistance development

• Combination Therapy Assessment:

  • Experimental design:

    • Group 1: Control IgG

    • Group 2: PD-1 mAb alone

    • Group 3: SFRP2 mAb alone

    • Group 4: Combination PD-1 mAb + SFRP2 mAb

  • Studies have shown SFRP2 mAb combination with PD-1 mAb had an additive antimetastatic effect when PD-1 mAb alone was minimally effective

• Immune Profiling:

  • Flow cytometry to assess changes in:

    • CD38 levels in tumor-infiltrating lymphocytes

    • PD-1 levels in T-cells

    • T-cell function and exhaustion markers

  • Combined with SFRP2 antibody-based detection methods

These methodologies help elucidate SFRP2's role in therapy resistance and identify strategies to overcome it.

How do different types of SFRP2 antibodies compare in research applications?

Various SFRP2 antibody formats offer distinct advantages for different research applications:

When selecting the optimal SFRP2 antibody format, researchers should consider:

• Target region specificity (internal region, C-terminal, etc.)

• Host species compatibility with experimental design

• Detection method requirements

• Application-specific sensitivity needs

What considerations are important when comparing results from studies using different SFRP2 antibodies?

When comparing studies using different SFRP2 antibodies, researchers should carefully evaluate:

• Epitope Targeting:

  • Antibodies targeting different regions of SFRP2 (e.g., AA 195-295 vs. AA 263-292) may yield varying results

  • Functional domains of SFRP2 include the cysteine-rich domain and netrin-like domain

• Methodology Differences:

  • Antigen retrieval protocols vary between antibodies

  • Different detection systems (direct HRP vs. indirect methods)

  • Varying sensitivities affect minimum detectable concentrations

• Cross-reactivity Profiles:

  • Some antibodies may detect both SFRP1 and SFRP2

  • Species cross-reactivity (human vs. mouse vs. rat) varies between antibodies

• Validation Standards:

  • Evaluate whether antibodies were validated using:

    • Knockout controls

    • Recombinant protein standards

    • Multiple application testing

These considerations ensure proper interpretation when comparing results across studies using different SFRP2 antibody reagents.