SFRP2 Antibody, FITC conjugated

What is SFRP2 and what are its primary biological functions?

SFRP2 (secreted frizzled-related protein 2) is a secreted glycoprotein containing an N-terminal cysteine-rich domain that shares 30-50% identity with the Wnt-binding site of frizzled receptors, and a C-terminal heparin-binding domain with weak homology to netrins. This protein plays critical roles in multiple cellular processes including embryogenesis, apoptosis regulation, and cell differentiation .

Functionally, SFRP2 has been identified as a key stem cell paracrine factor that mediates myocardial survival and repair following ischemic injury. It exerts its cardioprotective effects primarily by modulating Wnt signaling pathways, increasing cellular β-catenin levels, and upregulating antiapoptotic gene expression in cardiomyocytes . Additionally, methylation of the SFRP2 gene has been identified as a potential marker for colorectal cancer, indicating its relevance in oncological research .

How does SFRP2 interact with the Wnt signaling pathway?

SFRP2 modulates Wnt signaling through multiple mechanisms:

• Competitive inhibition: SFRP2 can compete with frizzled receptors for Wnt ligands by directly binding to Wnts, thereby preventing activation of canonical Wnt signaling .

• β-catenin modulation: Research indicates that SFRP2 increases both total cellular and nuclear β-catenin levels in cardiomyocytes, which has been demonstrated to protect neonatal rat cardiomyocytes against hypoxia/reoxygenation-induced apoptosis .

• Antagonism of proapoptotic Wnt3a: Studies have shown that the canonical Wnt, Wnt3a, is upregulated in ischemic cardiomyocytes and induces apoptosis. Importantly, SFRP2 blocks this proapoptotic effect of Wnt3a in vitro .

• Downstream target regulation: Gene expression analysis has identified Axin2 as a downstream target of SFRP2, the expression of which is activated by SFRP2 but inhibited by therapeutic interventions targeting SFRP2 .

Interestingly, under certain circumstances, SFRP2 may potentiate Wnt signaling by directly binding to frizzled receptors, similar to what has been observed with SFRP1 .

What are the optimal conditions for immunofluorescence applications using fluorescently-conjugated SFRP2 antibodies?

When utilizing fluorescently-conjugated SFRP2 antibodies for immunofluorescence applications, researchers should consider these optimal conditions:

For CoraLite® Plus 488 conjugated antibodies (similar to FITC), the excitation/emission maxima are approximately 493 nm/522 nm . It's essential to titrate the antibody in each specific experimental system to determine optimal dilution, as this can vary significantly depending on the sample type and preparation method.

What controls should be included when using fluorescently-labeled SFRP2 antibodies?

A robust experimental design for fluorescently-labeled SFRP2 antibody applications should include:

Positive Controls:

• Cell lines with known SFRP2 expression (e.g., SH-SY5Y cells)

• Tissues with documented SFRP2 expression (e.g., cardiac tissue)

• Recombinant SFRP2 protein

Negative Controls:

• Isotype control antibody (matching the SFRP2 antibody class, e.g., Mouse IgG1)

• Primary antibody omission

• Tissues from SFRP2 knockout models (when available)

Technical Controls:

• Unstained samples to assess autofluorescence

• Secondary antibody-only controls (if using indirect methods)

• Single-color controls when performing multiplex immunofluorescence

These controls help distinguish specific SFRP2 staining from non-specific binding or background fluorescence, which is particularly important in tissues with high autofluorescence such as cardiac tissue.

How should SFRP2 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of fluorescently-conjugated SFRP2 antibodies is critical for maintaining their activity and performance:

When working with these antibodies:

• Minimize freeze-thaw cycles

• Briefly centrifuge vials before opening to collect liquid

• Use aseptic technique to prevent contamination

• Return to -20°C promptly after use

• Protect from prolonged light exposure during all handling steps

How can SFRP2 antibodies be used to investigate cardiac repair mechanisms?

SFRP2 antibodies provide valuable tools for investigating cardiac repair mechanisms through multiple experimental approaches:

• Expression profiling: Fluorescently-conjugated SFRP2 antibodies can track SFRP2 expression patterns in cardiac tissue following ischemic injury, revealing spatial and temporal changes during the repair process.

• Pathway analysis: Co-localization studies with SFRP2 antibodies and markers of Wnt signaling (e.g., β-catenin) can elucidate how SFRP2 modulates this pathway in cardiac repair.

• Apoptosis studies: Research has shown that SFRP2 upregulates antiapoptotic genes like Birc1b in hypoxic cardiomyocytes. SFRP2 antibodies can be used to correlate SFRP2 expression with apoptotic markers to understand its protective mechanisms .

• Therapeutic targets: SFRP2 antibody-based blockade has demonstrated therapeutic potential in cardiomyopathic hamsters, increasing left ventricular ejection fraction from 40% to 49%, while reducing myocardial fibrosis by approximately 50% and decreasing apoptosis by about 65% .

• Fibrosis assessment: SFRP2 antibodies can help investigate the relationship between SFRP2 expression and cardiac fibrosis, particularly since SFRP2 blockade has been shown to increase the activity of matrix metalloproteinase-2, consistent with attenuated fibrosis .

What is the role of SFRP2 in stem cell-mediated tissue repair?

SFRP2 plays a critical role in stem cell-mediated tissue repair, particularly in cardiac regeneration:

• Paracrine mediator: SFRP2 has been identified as the key stem cell paracrine factor mediating the tissue repair effects of Akt-modified mesenchymal stem cells (Akt-MSCs) after ischemic injury .

• Akt-dependent expression: SFRP2 expression is dramatically upregulated (100-fold) in Akt-MSCs compared to control MSCs, and its expression/secretion depends on the PI3-kinase/Akt pathway .

• Prosurvival effects: SFRP2 confers significant prosurvival effects on hypoxic cardiomyocytes. Knockdown of SFRP2 expression results in attenuation of the prosurvival action of Akt-MSC-conditioned medium .

• Angiogenic properties: SFRP2 blockade increases myocardial levels of VEGF and hepatocyte growth factor (HGF), promoting angiogenesis which supports tissue repair processes .

• Targeted therapy potential: The understanding of SFRP2's role in stem cell-mediated repair has led to the development of antibody-based SFRP2 blockade as a potential antifibrotic therapy, highlighting the translational potential of this research .

How does SFRP2 influence gene expression in target cells?

SFRP2 modulates gene expression in target cells through several mechanisms:

• Antiapoptotic gene upregulation: Microarray analysis of hypoxic cardiomyocytes treated with SFRP2 revealed upregulation of Birc1b, an antiapoptotic gene belonging to the neuronal apoptosis inhibitory protein family .

• Axin2 activation: Gene expression analysis of hamster hearts and cultured fibroblasts identified Axin2 as a downstream target, the expression of which is activated by SFRP2 .

• β-catenin stabilization: SFRP2 increases total and nuclear β-catenin within hypoxic cardiomyocytes in a dose-dependent manner, potentially influencing the expression of β-catenin target genes .

• Cytochrome C regulation: Preliminary studies showed decreased mitochondrial cytochrome C levels in response to SFRP2 treatment, suggesting effects on the mitochondrial apoptotic pathway .

• Growth factor regulation: SFRP2 blockade increased myocardial levels of VEGF and hepatocyte growth factor (HGF), indicating that SFRP2 may normally suppress the expression of these angiogenic factors .

Why might I observe different molecular weights for SFRP2 in Western blots?

Discrepancies between theoretical and observed molecular weights of SFRP2 are common and should be considered during experimental design and data interpretation:

This 4 kDa difference may be attributed to:

• Post-translational modifications: SFRP2 is described as a glycoprotein, and glycosylation can significantly affect electrophoretic mobility .

• Protein processing: Proteolytic cleavage or other processing events may occur after translation.

• Tertiary structure effects: Unusual protein folding or amino acid composition can affect mobility in SDS-PAGE.

• Experimental conditions: Buffer composition, gel percentage, and running conditions can all influence apparent molecular weight.

When validating new SFRP2 antibodies, researchers should be aware of this discrepancy and not rely solely on the theoretical molecular weight for identification.

How can I optimize multiplex immunofluorescence experiments involving SFRP2 antibodies?

Successful multiplex immunofluorescence experiments with SFRP2 antibodies require careful consideration of several factors:

• Spectral compatibility: For fluorescently-conjugated SFRP2 antibodies similar to CoraLite Plus 488 (excitation/emission: 493nm/522nm), select additional fluorophores with minimal spectral overlap.

• Sequential vs. simultaneous staining: Determine whether sequential or simultaneous staining produces better results, particularly when examining proteins that might co-localize with SFRP2.

• Antibody validation: Validate each antibody individually before combining in multiplex experiments.

• Cross-reactivity prevention: Select antibodies from different host species or different isotypes when using secondary antibodies.

• Appropriate controls:

  • Single-color controls to assess spectral bleed-through

  • Isotype controls for each antibody

  • Fluorescence minus one (FMO) controls to establish gating boundaries

• Image acquisition settings: Optimize exposure times and detector gains for each fluorophore individually to prevent oversaturation while maintaining sufficient signal detection.

• Spectral unmixing: Consider using spectral unmixing algorithms during image analysis to separate overlapping fluorescence signals.

How do ischemic conditions affect SFRP2 expression and antibody detection?

Ischemic conditions significantly influence SFRP2 expression and may affect antibody detection:

• Expression changes: Research indicates that Wnt3a is upregulated in ischemic cardiomyocytes in vitro, suggesting that ischemia alters the Wnt signaling pathway, which could influence SFRP2 expression or localization .

• Prosurvival role: SFRP2 exerts prosurvival effects on hypoxic cardiomyocytes, suggesting its functional importance under ischemic conditions .

• Therapeutic implications: Antibody-based SFRP2 blockade showed functional improvement in cardiomyopathic hamsters, with a ~50% reduction in myocardial fibrosis, ~65% decrease in apoptosis, and ~75% increase in wall thickness, highlighting the role of SFRP2 in cardiac pathology .

• Methodological considerations:

  • Ischemic tissues may have altered autofluorescence properties

  • Protein degradation in ischemic regions may affect epitope availability

  • Increased background staining due to tissue damage and non-specific binding

  • Potential for hypoxia-induced changes in post-translational modifications affecting antibody recognition

Researchers should include appropriate ischemic and non-ischemic control tissues when designing experiments investigating SFRP2 in cardiovascular pathologies.

What are the emerging therapeutic applications for SFRP2 antibodies?

SFRP2 antibodies have shown promising therapeutic potential, particularly in cardiac disease:

• Antifibrotic therapy: Antibody-based SFRP2 blockade significantly improved cardiac function in cardiomyopathic hamsters, increasing left ventricular ejection fraction from 40% to 49%, while control groups showed further decline .

• Fibrosis reduction: SFRP2 antibody administration resulted in approximately 50% reduction in myocardial fibrosis, suggesting a direct antifibrotic effect .

• Anti-apoptotic effects: Treatment with SFRP2 antibodies led to approximately 65% decrease in apoptosis in cardiac tissue .

• Structural improvement: Wall thickness increased by approximately 75% following SFRP2 antibody treatment in cardiomyopathic hamsters .

• Matrix remodeling: Both mesenchymal stem cell therapy and SFRP2 antibody administration significantly increased the activity of myocardial matrix metalloproteinase-2, consistent with reduced fibrosis .

• Angiogenesis promotion: SFRP2 blockade increased myocardial levels of VEGF and hepatocyte growth factor (HGF), promoting angiogenesis .

These findings highlight the potential of SFRP2 as a specific target for antifibrotic therapy in cardiac disease and potentially other fibrotic disorders.

What future research directions should be explored with SFRP2 antibodies?

Several promising research directions warrant further investigation using SFRP2 antibodies:

• Mechanism elucidation: Further studies to clarify the precise molecular mechanisms by which SFRP2 modulates Wnt signaling in different cellular contexts.

• Tissue-specific effects: Investigation of SFRP2 functions in tissues beyond the heart, particularly in other fibrotic diseases.

• Therapeutic optimization: Refinement of antibody-based approaches targeting SFRP2 for potential clinical applications, including dosing strategies and delivery methods.

• Biomarker development: Exploration of SFRP2 as a biomarker for disease progression or treatment response in cardiac pathologies.

• Interaction studies: Further characterization of SFRP2 interactions with other proteins in the Wnt pathway and beyond.

• Post-translational modifications: Investigation of how glycosylation and other modifications affect SFRP2 function and antibody recognition.

• Combinatorial approaches: Testing SFRP2 antibody therapies in combination with other treatments for synergistic effects in cardiac repair.

• Genetic variation studies: Examination of how genetic variants in SFRP2 affect protein function and response to antibody-based therapies.