SFRP5 Antibody, Biotin conjugated

What is SFRP5 and what are its primary biological functions?

SFRP5 (Secreted frizzled-related protein 5) functions as an adipokine that acts as an inhibitor of the noncanonical WNT signaling pathway. Research indicates it exerts anti-inflammatory and insulin-sensitizing effects, although some contradictory findings have been reported. SFRP5 is implicated in metabolic regulation, with studies showing positive correlations between circulating SFRP5 levels and insulin sensitivity measured via hyperinsulinemic-euglycemic clamp techniques . The protein exists with several aliases including Frizzled-related protein 1b (FRP-1b) and Secreted apoptosis-related protein 3 (SARP-3) . SFRP5's relationship with insulin sensitivity appears to be primarily dependent on adiponectin, with research demonstrating significant positive correlation between serum levels of these proteins (r = 0.55, p < 0.001) .

What are the key specifications of commercial SFRP5 Antibody, Biotin conjugated preparations?

Commercial SFRP5 Antibody, Biotin conjugated preparations are typically polyclonal antibodies derived from rabbit hosts immunized with recombinant human SFRP5 protein fragments (specifically amino acids 169-261). These preparations undergo Protein G purification with purity levels exceeding 95%. The antibodies are supplied in liquid form containing preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol in 0.01M PBS, pH 7.4). They require storage at -20°C or -80°C, with repeated freeze-thaw cycles being detrimental to antibody integrity. The biotin conjugation enables various detection strategies in immunoassays without requiring secondary antibody conjugates .

How does SFRP5 integrate into the broader Wnt signaling pathway framework?

SFRP5 functions as a regulatory component within the Wnt signaling network, primarily as an inhibitor of the noncanonical Wnt pathway. Research demonstrates that SFRP5 interacts directly with Wnt5a, but surprisingly, this interaction does not always result in inhibition of Wnt5a activity. In rheumatoid arthritis tissue-derived fibroblast-like synoviocytes (td-FLS), the combination of SFRP5 with Wnt5a actually enhances pro-inflammatory responses rather than suppressing them . Mechanistically, SFRP5 appears to influence canonical Wnt signaling through modulation of TCF4 expression. Studies have observed that treatment with Wnt5a alone or in combination with SFRP5 decreases expression of the canonical transcription factor TCF4 and reduces protein levels of the canonical coreceptor LRP5 . This suggests that SFRP5's role in Wnt signaling is context-dependent and may involve complex cross-talk between canonical and noncanonical pathways.

What are the optimal applications and detection methods for SFRP5 Antibody, Biotin conjugated?

SFRP5 Antibody, Biotin conjugated is primarily optimized for Enzyme-Linked Immunosorbent Assay (ELISA) applications, though its biotin conjugation makes it versatile for multiple immunodetection platforms. The antibody demonstrates human species reactivity and is derived from rabbit hosts, making it compatible with anti-rabbit secondary detection systems. For optimal detection sensitivity, researchers should implement streptavidin-based detection systems (HRP-streptavidin or fluorophore-conjugated streptavidin) that leverage the high-affinity biotin-streptavidin interaction. Buffer conditions should maintain pH 7.4 as specified in the product formulation. Importantly, all applications should follow the vendor's storage recommendations (-20°C or -80°C) to preserve activity, with aliquoting advisable to minimize freeze-thaw cycles . When designing experimental controls, consider including both negative controls (non-specific rabbit IgG) and positive controls (recombinant human SFRP5) to validate specificity and performance.

How should researchers design experiments to investigate SFRP5's influence on inflammatory processes?

Based on current research methodologies, a comprehensive experimental design for investigating SFRP5's role in inflammation should implement multiple approaches. Researchers should begin with cell culture systems relevant to the inflammatory context being studied (e.g., fibroblast-like synoviocytes for rheumatoid arthritis research). Cells should be treated with recombinant Wnt5a (300 ng/ml) alone and in combination with recombinant SFRP5, with appropriate timing (adding SFRP5 10 minutes before Wnt5a) . Analysis should include:

• Gene expression profiling via qPCR for inflammatory markers (IL1β, IL8, IL6, CCL2, COX2, CXCL10) at multiple time points (4h and 24h post-stimulation)

• Protein expression analysis via Western blotting and ELISA for both signaling mediators and secreted inflammatory factors

• Pathway analysis examining both canonical (β-catenin, TCF4, LRP5) and noncanonical Wnt components

This approach enabled researchers to identify the surprising finding that SFRP5 enhances rather than inhibits Wnt5a-induced inflammatory responses in specific cellular contexts . Controls should include untreated cells and treatments with individual factors to establish baseline responses.

What protocols are recommended for isolating and culturing cells to study SFRP5 function?

Based on established methodologies for studying SFRP5 in inflammatory contexts, researchers should consider three primary cell isolation approaches:

For tissue-derived fibroblast-like synoviocytes (td-FLS):

• Obtain fresh synovial tissue specimens through surgical procedures

• Process using established mechanical digestion and enzyme treatment protocols

• Culture in DMEM with 10% FBS and antibiotics

• Use passages 3-8 for experimental procedures to ensure phenotypic stability

For fluid-derived fibroblast-like synoviocytes (fd-FLS):

• Centrifuge synovial fluid at 1000 rpm for 30 minutes

• Resuspend cell pellets in DMEM containing 20% FBS and antibiotics

• After 72 hours, remove non-adherent cells and continue culture

• Utilize passage 3 cells for RNA and protein extraction experiments

For fibrocyte isolation:

• Mix peripheral blood with DMEM (2:1 ratio)

• Layer over Ficoll/Plaque and centrifuge at 1800 rpm for 30 minutes

• Collect peripheral blood mononuclear cells (PBMCs) and culture in DMEM with 20% FBS

• Replace medium on day 5 and continue culture for 14 days

• Identify fibrocytes by their elongated spindle-shaped morphology and oval nuclei

These protocols enable comprehensive analysis of SFRP5 function across multiple relevant cell types in inflammatory disease contexts.

How does SFRP5 relate to insulin sensitivity and metabolic regulation?

Research utilizing hyperinsulinemic-euglycemic clamp techniques has established a significant positive correlation between circulating SFRP5 levels and insulin sensitivity (r = 0.23, p = 0.006). Studies comparing normal-weight and overweight/obese subjects demonstrate lower baseline SFRP5 in overweight/obese individuals (p = 0.01). Multiple regression analysis reveals that adiponectin is independently associated with SFRP5, suggesting interconnected regulatory pathways .

Experimental manipulations of insulin and free fatty acid (FFA) levels yield important insights into SFRP5 regulation. Insulin infusion decreases serum SFRP5 levels at both 120 minutes (p = 0.02) and 360 minutes (p = 0.031). Intriguingly, this insulin-induced reduction is abolished when Intralipid/heparin (raising FFA levels) is co-administered. This demonstrates a complex regulatory relationship where insulin appears more influential than FFA in controlling circulating SFRP5 . These findings suggest SFRP5 may serve as a biomarker for metabolic health and provide mechanistic insights into insulin-adipokine interactions.

What is the relationship between SFRP5 and Wnt5a in inflammatory conditions like rheumatoid arthritis?

Contrary to initial hypotheses predicting anti-inflammatory effects, research has revealed that SFRP5 enhances rather than inhibits Wnt5a-induced inflammation in rheumatoid arthritis tissue-derived fibroblast-like synoviocytes (RA td-FLS). Quantitative PCR analysis demonstrates that Wnt5a stimulation (300 ng/ml) induces significant upregulation of pro-inflammatory mediators in RA td-FLS, particularly IL1β (>48-fold increase), IL8 (25.7-fold), IL6 (13.7-fold), CCL2 (8.5-fold), and COX2 (7.4-fold) after 4 hours .

Surprisingly, when SFRP5 is added prior to Wnt5a treatment, the inflammatory response is further amplified, with even higher expression of IL1β (65.5-fold), IL8 (45.2-fold), IL6 (30.1-fold), and COX2 (24.2-fold). This synergistic effect appears linked to inhibition of canonical Wnt signaling, as evidenced by decreased expression of TCF4 and reduced protein levels of the canonical coreceptor LRP5 following combined Wnt5a/SFRP5 treatment . These findings challenge the conventional understanding of SFRP5 as purely anti-inflammatory and highlight the context-dependent nature of Wnt signaling modulation in disease states.

How does SFRP5 expression compare across different cell types involved in rheumatoid arthritis?

Research examining SFRP5 expression patterns across multiple cell types implicated in rheumatoid arthritis reveals consistent expression profiles. Quantitative PCR analysis demonstrates that tissue-derived fibroblast-like synoviocytes (td-FLS), fluid-derived fibroblast-like synoviocytes (fd-FLS), and fibrocytes from rheumatoid arthritis patients express similar levels of both Wnt5a and a set of Wnt5a receptors/coreceptors . This consistent expression pattern across cell types suggests that SFRP5-mediated signaling may be a conserved mechanism within the joint microenvironment during inflammatory arthritis.

The comparable expression levels across these cell types, which represent different stages of cellular differentiation and tissue localization, indicates that Wnt5a signaling elements may be fundamental to the pathophysiology of rheumatoid arthritis rather than restricted to specific cellular compartments. This finding provides important context for interpreting intervention studies targeting the Wnt5a pathway and suggests that therapeutic approaches may need to consider effects across multiple cell types within the joint microenvironment .

How should researchers address contradictory findings regarding SFRP5's role in inflammatory conditions?

When confronting contradictory findings regarding SFRP5's inflammatory effects, researchers should implement a systematic approach:

• Consider cellular context: Evidence indicates SFRP5's effects are highly context-dependent. For example, while SFRP5 is generally considered anti-inflammatory, it enhances Wnt5a-induced inflammation in RA td-FLS . Similar observations were reported in macrophages, where SFRP5 increased IL12, IL6, and TNF levels when combined with Wnt5a .

• Examine pathway interactions: Contradictions may stem from cross-talk between signaling pathways. Research shows SFRP5 influences canonical Wnt signaling by decreasing TCF4 expression, creating a direct link between pro-inflammatory effects and canonical pathway inhibition .

• Standardize experimental conditions: Variations in SFRP5 concentration, timing of administration relative to other factors (like Wnt5a), and incubation periods significantly impact observed effects. Standardizing the protocol where SFRP5 is added 10 minutes before Wnt5a and measuring responses at multiple timepoints (4h and 24h) helps resolve discrepancies .

• Validate at protein level: Many contradictions arise from differences between mRNA expression and protein secretion. Researchers should complement gene expression studies with protein-level analyses in cell supernatants to establish functional relevance .

By addressing these factors methodically, researchers can better interpret seemingly contradictory findings and develop more nuanced understanding of SFRP5's complex biological roles.

What controls are essential when using SFRP5 Antibody, Biotin conjugated in experimental settings?

When utilizing SFRP5 Antibody, Biotin conjugated in research applications, establishing rigorous controls is essential for valid data interpretation:

• Isotype controls: Include rabbit IgG at equivalent concentrations to the SFRP5 antibody to identify non-specific binding, particularly important given the polyclonal nature of the antibody .

• Antigen blocking controls: Pre-incubate the antibody with recombinant human SFRP5 protein (specifically amino acids 169-261, as this region was used as the immunogen) to confirm binding specificity .

• Positive tissue/cell controls: Include samples known to express SFRP5 (adipose tissue or cultured adipocytes) as positive controls for staining procedures or immunoassays.

• Negative tissue/cell controls: Utilize tissues or cell lines with minimal SFRP5 expression to establish background signal thresholds.

• Signal amplification controls: When using streptavidin-based detection systems, include samples treated with streptavidin reagents alone (without primary antibody) to identify non-specific interactions of the detection system.

• Cross-reactivity assessment: If working with non-human samples, despite the antibody being raised against human SFRP5, validate detection specificity through Western blotting or ELISA with species-specific recombinant proteins.

These controls collectively ensure that signals detected are specifically attributable to SFRP5 rather than experimental artifacts or cross-reactivity.

How can researchers address temporal variations in SFRP5-mediated responses?

Research has demonstrated significant temporal dynamics in SFRP5-mediated inflammatory responses that require careful experimental design and interpretation. When studying SFRP5 effects, particularly in combination with Wnt5a, researchers should implement the following approaches:

These approaches collectively enable researchers to capture the complex temporal dynamics of SFRP5-mediated responses and avoid misleading interpretations based on single-timepoint data.

What are the molecular mechanisms through which SFRP5 modulates Wnt signaling pathways?

In rheumatoid arthritis tissue-derived fibroblast-like synoviocytes, SFRP5 enhances rather than inhibits Wnt5a-induced inflammation. Mechanistically, this occurs through inhibition of canonical Wnt signaling components, as evidenced by decreased gene expression of TCF4 and reduced protein levels of the canonical coreceptor LRP5 following combined Wnt5a/SFRP5 treatment . This observation aligns with previous findings that noncanonical Wnt signaling mechanisms can inhibit the β-catenin pathway by increasing β-catenin turnover or decreasing β-catenin/TCF association with DNA .

Interestingly, while SFRP5 affects TCF4 expression, studies have not observed changes in β-catenin expression after stimulation, consistent with research showing that Wnt5a inhibits canonical Wnt signaling at the level of TCF transcription rather than β-catenin levels . These findings collectively suggest that SFRP5 functions through complex pathway cross-talk rather than simple inhibition of Wnt ligand binding.

How do insulin and free fatty acids regulate circulating SFRP5 levels?

Advanced metabolic research utilizing hyperinsulinemic-euglycemic clamp techniques has revealed sophisticated regulatory mechanisms controlling circulating SFRP5 levels. Insulin administration demonstrates a significant suppressive effect on serum SFRP5 concentrations, with statistically significant decreases observed both at 120 minutes (p = 0.02) and with sustained suppression at 360 minutes (p = 0.031) .

This insulin-mediated suppression exhibits complex interaction with lipid metabolism. When insulin is administered concurrently with Intralipid/heparin infusion (which elevates free fatty acid levels), the suppressive effect of insulin on SFRP5 is completely abolished. Importantly, Intralipid/heparin infusion alone does not significantly alter SFRP5 levels, indicating that insulin is the primary regulatory factor, with FFA serving as a modulator of insulin's effects rather than directly regulating SFRP5 .

The mechanistic basis for this interaction remains incompletely understood but may involve insulin-responsive elements in SFRP5 gene regulatory regions or post-translational modifications affecting protein stability and secretion. This regulatory relationship has potential implications for understanding how metabolic dysregulation in conditions like insulin resistance and dyslipidemia might impact SFRP5-mediated signaling pathways in tissues.

What emerging research directions might advance our understanding of SFRP5 biology and its therapeutic potential?

Several promising research directions could significantly advance understanding of SFRP5 biology and its therapeutic applications:

• Tissue-specific knockout models: Developing conditional knockout models targeting SFRP5 in specific tissues (adipose, synovium, liver) would help delineate tissue-specific functions and potential compensatory mechanisms that explain contradictory findings across studies.

• Post-translational modification analysis: Research investigating how glycosylation, phosphorylation, and other modifications affect SFRP5 bioactivity could explain context-dependent effects observed in different experimental systems.

• Receptor interaction profiling: Comprehensive analysis of SFRP5 interactions with various Frizzled receptors and co-receptors would clarify its selectivity and contextual signaling outcomes. Current research indicates expression of multiple Wnt5a receptors/coreceptors across relevant cell types .

• Development of selective SFRP5 mimetics: Creating peptide fragments or small molecules that selectively mimic SFRP5's beneficial metabolic effects while avoiding pro-inflammatory activities would advance therapeutic applications.

• Single-cell transcriptomics in disease tissues: Applying these technologies to tissues from conditions like rheumatoid arthritis would identify cell populations most responsive to SFRP5 signaling and clarify heterogeneous responses.

• Expanded protein-level analysis: Future studies should complement gene expression data with comprehensive protein secretion analysis to address limitations noted in current research .

These approaches would address current knowledge gaps, particularly regarding the discrepancy between SFRP5's reported anti-inflammatory effects in some contexts and pro-inflammatory actions in others, potentially leading to more precisely targeted therapeutic interventions.