SFRP2 Antibody, Biotin conjugated

What is SFRP2 and why is it an important research target?

SFRP2 (Secreted frizzled-related protein 2) is a secreted glycoprotein that functions as a modulator of Wnt signaling through direct interaction with Wnt proteins. It plays significant roles in regulating cell growth and differentiation in specific cell types . SFRP2 has been implicated in diverse cellular processes, including embryogenesis, regulation of cell apoptosis, and cell differentiation . It contains an N-terminal cysteine-rich domain (30-50% identical to the Wnt-binding site of frizzled receptors) and a C-terminal heparin-binding domain with weak homology to netrins .

Research interest in SFRP2 stems from its involvement in multiple pathological conditions. For instance, SFRP2 promotes metastatic osteosarcoma cell migration and tumor angiogenesis . Additionally, methylation of this gene has been investigated as a potential marker for colorectal cancer . Recent studies have also shown that SFRP2 regulates the WNT/β-catenin pathway, influencing the development of conditions such as aldosterone-producing adenoma .

What are the optimal applications for biotin-conjugated SFRP2 antibodies?

Biotin-conjugated SFRP2 antibodies are particularly valuable for several laboratory techniques:

• ELISA (Enzyme-Linked Immunosorbent Assay): The biotin conjugation allows for high-sensitivity detection with avidin/streptavidin-HRP systems, with recommended dilutions of approximately 1:10,000 .

• Western Blot (WB): Biotin-conjugated antibodies enable sensitive protein detection, typically used at dilutions of 1:500 .

• Immunoprecipitation (IP): These antibodies perform well in pulling down SFRP2 at dilutions around 1:200 .

The biotin-conjugation provides advantages in detection sensitivity and signal amplification compared to unconjugated antibodies, making them particularly useful for detecting low-abundance SFRP2 in complex biological samples .

What is the molecular weight and expression pattern of SFRP2 protein?

When working with SFRP2 antibodies, researchers should expect to detect:

• Calculated molecular weight: 33.5-34 kDa (295 amino acids)

• Observed molecular weight: Typically 33-38 kDa in Western blot applications

SFRP2 exhibits a specific tissue expression pattern relevant for experimental design and control selection:

• High expression: Adipose tissue, small intestine, and colon

• Moderate expression: Heart, brain, skeletal muscle, pancreas, thymus, prostate, testis, and ovary

This expression profile should be considered when selecting appropriate positive control tissues for antibody validation and experimental design .

How should biotin-conjugated SFRP2 antibody be used in sandwich ELISA protocols?

For optimal results in sandwich ELISA using biotin-conjugated SFRP2 antibody, follow this methodological approach:

• Plate preparation: Coat microtiter wells with a capture antibody specific to human SFRP2 (pre-coated plates are available in commercial kits) .

• Sample addition: Add 100 μL of standards or samples to appropriate wells and incubate at 37°C for 80 minutes .

• Washing step: Discard well contents and wash with 200 μL 1× Wash Buffer (3 times) .

• Biotin antibody addition: Add 100 μL of diluted biotin-conjugated SFRP2 antibody (typically at 1:10,000 dilution) and incubate at 37°C for 50 minutes .

• Wash step: Repeat washing procedure as above .

• Detection step: Add 100 μL of Streptavidin-HRP Working Solution (1×) and incubate at 37°C for 50 minutes .

• Final washing: Wash 5 times with 200 μL 1× Wash Buffer .

• Substrate reaction: Add 90 μL TMB Substrate Solution, incubate at 37°C for 20 minutes in the dark .

• Termination and detection: Add 50 μL Stop Solution, shake plate for 1 minute to mix, and record OD at 450 nm immediately .

This protocol yields high sensitivity with reported intra-assay precision (CV% < 8%) and inter-assay precision (CV% < 10%) . Recovery rates in serum, EDTA plasma, and heparin plasma range from 78-97% .

What validation tests should be performed when using biotin-conjugated SFRP2 antibody?

To ensure reliable results with biotin-conjugated SFRP2 antibody, implement these validation steps:

• Positive control testing: Use tissues/cells known to express SFRP2, such as heart tissue (mouse or rat) or A549 cells, as recommended by antibody manufacturers .

• Western blot validation: Verify single band detection at ~33-38 kDa in positive control samples. For example, human cell lines such as Hela, A431, Caco-2, and U-87MG have been used successfully .

• Specificity testing: Compare results in samples with confirmed SFRP2 expression versus negative controls. Recommended positive samples include human Caco-2 cells for flow cytometry applications .

• Dilution linearity assessment: Test antibody performance across multiple dilutions (1:2, 1:4, 1:8, 1:16) in various matrices (serum, EDTA plasma, heparin plasma) to establish optimal working concentrations .

• Cross-reactivity evaluation: Test with human, mouse, and rat samples to confirm species reactivity as indicated by the manufacturer .

Include appropriate controls in each experiment, and document lot-specific validation results to ensure experimental reproducibility and reliability across studies.

What are optimal storage conditions for maintaining biotin-conjugated SFRP2 antibody activity?

To preserve antibody integrity and ensure consistent experimental outcomes:

• Long-term storage: Maintain at -20°C for optimal stability . Avoid repeated freeze-thaw cycles by preparing working aliquots.

• Buffer composition: Most biotin-conjugated SFRP2 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Concentration: Typically provided at 0.64-0.72 μg/μl in antibody stabilization buffer .

• Aliquoting recommendations: Manufacturers note that aliquoting is unnecessary for -20°C storage, though it may be beneficial for antibodies frequently accessed .

• Working solution stability: Once diluted, use within the same day for optimal performance in assays such as ELISA and Western blotting.

When handling biotin-conjugated antibodies, avoid prolonged exposure to light, as this may affect the biotin moiety and reduce detection sensitivity in downstream applications.

How can biotin-conjugated SFRP2 antibody be used to study SFRP2's role in cancer progression?

Biotin-conjugated SFRP2 antibodies offer valuable methodological approaches for investigating SFRP2's role in cancer:

• Metastasis investigation: Using immunohistochemistry or immunofluorescence, researchers can detect SFRP2 in cancer tissues to assess correlation with metastatic potential. For instance, studies have shown that SFRP2 promotes migration and invasion of metastatic osteosarcoma cells .

• Protein interaction studies: Biotin-conjugated antibodies can be utilized in co-immunoprecipitation assays to identify SFRP2-interacting proteins in cancer pathways . This is particularly relevant for studying SFRP2's interaction with WNT pathway components.

• Expression profiling: Western blot analysis of cancer cell lysates using biotin-conjugated SFRP2 antibody helps establish expression patterns across different cancer types and stages. Validated cell lines include Hela, A431, Caco-2, and U-87MG .

• Therapeutic target validation: In humanized monoclonal antibody development against SFRP2 for cancer treatment, biotin-conjugated antibodies can help validate target engagement through competitive binding assays .

Research has demonstrated that SFRP2-targeted immunotherapy with humanized monoclonal antibodies reduces metastatic osteosarcoma growth through direct antitumor effects, antiangiogenic activity, and immune system modulation . Biotin-conjugated antibodies provide tools to study these mechanisms in detail.

How is SFRP2 antibody used in exploring the relationship between SFRP2 and immune regulation?

Recent research demonstrates SFRP2's unexpected role in immune regulation, which can be studied using biotin-conjugated antibodies through these methodological approaches:

• T-cell function analysis: Flow cytometry with biotin-conjugated SFRP2 antibodies can detect SFRP2's influence on T-cell populations. Research has shown that SFRP2 affects T-cell proliferation and promotes NFATc3, CD38, and PD-1 expression in T-cells .

• Co-culture experiments: Biotin-conjugated antibodies can monitor SFRP2 levels in tumor cell/T-cell co-culture models, helping elucidate mechanisms of tumor-induced immunosuppression. Studies have demonstrated that humanized SFRP2 monoclonal antibodies rescue the suppression of T-cell proliferation in co-culture experiments .

• Signaling pathway investigation: Western blot analysis with biotin-conjugated SFRP2 antibodies helps track downstream signaling events. Research shows SFRP2 affects NFATc3 activation and CD38 expression in immune cells .

• PD-1 resistance mechanisms: Immunoprecipitation with biotin-conjugated SFRP2 antibodies can identify protein complexes involved in PD-1 inhibitor resistance. Studies indicate that blocking SFRP2 can overcome resistance to PD-1 inhibitors in metastatic osteosarcoma .

These applications underline SFRP2's emerging role as an immunomodulatory protein that affects T-cell function and response to checkpoint inhibitor therapy, highlighting the importance of SFRP2 antibodies in both cancer and immunology research .

What are common challenges when using biotin-conjugated SFRP2 antibody in Western blot applications?

Researchers frequently encounter these technical challenges when using biotin-conjugated SFRP2 antibody in Western blot:

• Multiple bands: SFRP2 may appear at different molecular weights (33-38 kDa range). This variation could represent:

  • Post-translational modifications

  • Different isoforms

  • Partial degradation products

  Solution: Validate with positive controls like human Hela, A431, Caco-2, or U-87MG whole cell lysates, which show bands at approximately 38 kDa .

• High background: Biotin-conjugated antibodies can produce elevated background due to endogenous biotin in samples.

  Solution: Use biotin blocking kits before antibody incubation and optimize blocking conditions (5% Non-fat Milk/TBS for 1.5 hours at room temperature has been validated) .

• Inconsistent signal intensity: Signal variations between experiments may occur.

  Solution: Standardize protein loading (50 μg per lane recommended), use consistent transfer conditions (150 mA for 50-90 minutes), and maintain consistent antibody concentration (0.25 μg/mL antibody concentration validated for detection) .

• Species-specific variations: Reactivity differences between human, mouse and rat samples may cause inconsistent results.

  Solution: Verify species cross-reactivity and optimize antibody concentration for each species separately .

• Detection sensitivity: Biotin-conjugation should enhance sensitivity, but some applications may require signal amplification.

  Solution: Use enhanced chemiluminescent detection (ECL) kits for optimal visualization, as validated in published protocols .

These troubleshooting approaches have been validated in experimental settings and can help ensure reliable Western blot results with biotin-conjugated SFRP2 antibodies.

How can specificity of biotin-conjugated SFRP2 antibody results be verified?

To confirm the specificity of results obtained with biotin-conjugated SFRP2 antibody, implement these methodological validation steps:

• Multiple detection methods: Verify findings using complementary techniques:

  • Western blot (WB): Confirms protein size at 33-38 kDa

  • Immunofluorescence (IF): Confirms cellular localization

  • Flow cytometry: Validates expression in cell populations

  Research shows consistent detection across these methods strengthens result reliability .

• Positive and negative controls:

  • Positive controls: Include tissues with known high SFRP2 expression (adipose tissue, small intestine, colon)

  • Negative controls: Use samples where SFRP2 expression is absent or minimal

  • Antibody controls: Include a non-specific IgG control of the same host species and concentration

• Blocking peptide competition: Pre-incubate antibody with the immunizing peptide (synthetic peptide within amino acid region 225-295 on human SFRP2) before application to samples. Signal disappearance confirms specificity.

• Orthogonal antibody validation: Compare results with a different antibody targeting another epitope of SFRP2.

• Genetic validation: When possible, correlate with knockdown/knockout models or samples with known genetic alterations affecting SFRP2 expression.

These approaches provide multiple lines of evidence supporting result specificity, enabling confident interpretation of SFRP2 detection data across experimental systems.

How are biotin-conjugated SFRP2 antibodies being used in WNT pathway research?

Biotin-conjugated SFRP2 antibodies serve as critical tools for investigating the complex interactions between SFRP2 and WNT signaling through these methodological approaches:

• WNT/β-catenin pathway analysis: Western blot with biotin-conjugated SFRP2 antibodies helps elucidate how SFRP2 regulates β-catenin expression. Research demonstrates that SFRP2 can inhibit the WNT/β-catenin signaling pathway by suppressing β-catenin expression, affecting conditions like aldosterone-producing adenoma .

• Co-immunoprecipitation studies: These antibodies enable investigation of SFRP2's direct interactions with Wnt proteins. SFRP2 functions as a modulator of Wnt signaling through direct interaction with Wnts, affecting cell growth and differentiation .

• Functional activity assessment: In combination with recombinant proteins, biotin-conjugated antibodies can assess SFRP2's impact on WNT pathway activity. SFRP2 has demonstrated multiple biochemical activities including endopeptidase activator activity, Wnt-activated receptor activity, and Wnt-protein binding .

• Tissue-specific WNT modulation: Immunohistochemistry with these antibodies reveals tissue-specific patterns of SFRP2 expression and correlation with WNT pathway components. This is particularly important as SFRP2 may be critical for eye retinal development and myogenesis .

• Cancer pathway studies: Flow cytometry combined with pathway inhibitors helps analyze how SFRP2 affects WNT signaling in cancer progression. Research shows SFRP2 promotes metastatic cell migration and angiogenesis, partly through WNT pathway modulation .

Understanding these SFRP2-WNT interactions has significant therapeutic implications, as both SFRP2 and WNT pathways are potential therapeutic targets for inhibiting pathological processes in multiple diseases .

What role does SFRP2 play in immunotherapy, and how can antibodies facilitate this research?

Emerging research reveals SFRP2's unexpected immunomodulatory functions that can be investigated using biotin-conjugated antibodies:

• PD-1 inhibitor resistance: Biotin-conjugated antibodies enable investigation of SFRP2's role in therapy resistance mechanisms. Studies reveal that humanized SFRP2 monoclonal antibody treatment can overcome resistance to PD-1 inhibitors in metastatic osteosarcoma models .

• T-cell function studies: These antibodies can track SFRP2's effects on T-cell phenotypes and functional properties. Research demonstrates that SFRP2 promotes NFATc3, CD38 and PD-1 expression in T-cells, while anti-SFRP2 antibody treatment counteracts these effects and increases NAD+ levels .

• Tumor microenvironment characterization: By analyzing SFRP2 expression in tumor-infiltrating lymphocytes, researchers can assess its impact on the immune microenvironment. Blocking SFRP2 with humanized monoclonal antibodies reduces CD38 levels in tumor-infiltrating lymphocytes and T-cells, as well as lowering PD-1 levels in T-cells .

• Combinatorial therapy development: Biotin-conjugated antibodies help evaluate SFRP2 targeting in combination with other immunotherapies. While PD-1 monoclonal antibody alone showed minimal effect in some models, combining it with anti-SFRP2 antibody demonstrated additive antimetastatic effects .

• Mechanism of action studies: These antibodies facilitate investigation of molecular pathways underlying SFRP2's immunomodulatory effects. Research indicates that SFRP2-targeted immunotherapy reduces metastatic osteosarcoma growth through multiple mechanisms: direct antitumor effects, antiangiogenic activity, and immune system modulation .

This research direction represents a significant paradigm shift, positioning SFRP2 as both a cancer progression factor and an immunomodulatory protein, with implications for developing novel combination immunotherapy approaches .