SFRP5 Antibody, FITC conjugated

What is SFRP5 and what are its primary biological functions?

SFRP5 (Secreted frizzled-related protein 5) functions as a critical modulator of Wnt signaling through direct interaction with Wnt proteins. It plays an essential role in regulating cell growth and differentiation in specific cell types and tissues. SFRP5 contains a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzled proteins, allowing it to act as a soluble modulator of Wnt signaling pathways .

In particular, SFRP5 appears to be involved in determining the polarity of photoreceptor cells in the retina, with high expression observed in the retinal pigment epithelium . Additionally, SFRP5 functions as an anti-inflammatory adipokine whose expression is altered in models of obesity and type 2 diabetes. Research indicates that it is secreted by adipocytes and controls the microenvironment of white adipose tissue under conditions of metabolic stress .

What is the molecular structure and characteristics of SFRP5?

The SFRP5 gene encodes a protein consisting of 317 amino acid residues structured into 3 coding exons . The fully processed protein has a calculated molecular weight of 36 kDa, though it is frequently observed at 36-42 kDa in experimental settings, likely due to post-translational modifications . The protein contains specific structural domains including a cysteine-rich domain that mediates Wnt interaction.

The human SFRP5 protein is identified by UniProt ID Q5T4F7, with corresponding IDs in mouse (Q9WU66) and rat systems . SFRP5 is also known by several aliases including Frizzled-related protein 1b (FRP-1b), Secreted apoptosis-related protein 3 (SARP-3), and sFRP-5, reflecting its discovery in different research contexts .

What considerations should guide selection of an SFRP5 antibody for specific applications?

When selecting an SFRP5 antibody for research, consider these critical factors:

• Species reactivity: Available antibodies show reactivity with human, mouse, and rat samples, but cross-reactivity varies between products . For instance, some antibodies are specifically designed for human SFRP5 detection (e.g., CSB-PA021141LC01HU) , while others demonstrate broader reactivity across species .

• Application compatibility: Different antibodies are validated for specific applications including Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF)/ICC, and ELISA . Antibody 14283-1-AP, for example, has been positively tested in multiple applications with specific recommended dilutions:

  • Western Blot: 1:500-1:1000

  • Immunohistochemistry: 1:50-1:500

  • Immunofluorescence: 1:200-1:800

• Immunogen information: The specific epitope or region used as immunogen affects specificity. For example, ab230425 uses a synthetic peptide within Human SFRP5 aa 150-250 , while CSB-PA021141LC01HU uses recombinant Human SFRP5 protein (169-261AA) .

• Conjugation requirements: For fluorescence applications, consider conjugated options like FITC-conjugated antibodies which eliminate secondary antibody requirements .

What are the specific characteristics of FITC-conjugated SFRP5 antibodies?

FITC-conjugated SFRP5 antibodies combine the specificity of SFRP5 recognition with the fluorescent properties of fluorescein isothiocyanate, enabling direct visualization in fluorescence-based applications. Key characteristics include:

• Conjugation chemistry: The antibody is directly labeled with FITC, which has an excitation maximum around 495 nm and emission maximum around 519 nm, producing green fluorescence.

• Storage requirements: These conjugated antibodies require careful storage at -20°C or -80°C to preserve both antibody integrity and fluorophore activity . Avoid repeated freeze-thaw cycles which can degrade both components.

• Buffer composition: Typically preserved in a buffer containing 0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4, which maintains antibody stability and fluorophore activity .

• Applications: Primarily used for direct immunofluorescence microscopy, flow cytometry, and other fluorescence-based detection methods without requiring secondary antibody incubation.

• Purity: Higher-quality antibodies undergo protein G purification with >95% purity to minimize non-specific binding and background fluorescence .

How should SFRP5 antibody be optimized for Western blot applications?

For optimal Western blot results with SFRP5 antibody, follow these methodological considerations:

• Sample preparation: Tissue lysates from heart, retina, and cell lines including HUVEC and L02 have demonstrated good SFRP5 detection . For tissue lysates, use approximately 35 μg of protein per lane as demonstrated with mouse heart tissue lysate .

• Expected band size: Look for bands at the predicted molecular weight of 36 kDa, though the observed molecular weight can range from 36-42 kDa due to post-translational modifications .

• Dilution optimization: Start with the recommended dilution range of 1:500-1:1000 for Western blot applications, but perform a dilution series (e.g., 1:250, 1:500, 1:1000, 1:2000) to determine optimal signal-to-noise ratio for your specific samples .

• Detection method: ECL (Enhanced Chemiluminescence) technique has been successfully employed for SFRP5 detection in Western blot applications .

• Controls: Include positive controls such as mouse heart tissue or retina tissue lysates where SFRP5 expression has been confirmed . For negative controls, consider using tissue or cell types with minimal SFRP5 expression or SFRP5 knockdown samples.

What protocol modifications are needed for FITC-conjugated SFRP5 antibody in immunofluorescence studies?

When using FITC-conjugated SFRP5 antibody for immunofluorescence applications:

• Direct detection protocol: Unlike unconjugated antibodies, FITC-conjugated antibodies allow for single-step detection without secondary antibody:

  • Fix and permeabilize cells/tissues using standard protocols (4% paraformaldehyde followed by 0.1-0.5% Triton X-100)

  • Block with appropriate blocking buffer (5% normal serum in PBS with 0.1% Tween-20)

  • Incubate with FITC-conjugated SFRP5 antibody at 1:200-1:800 dilution (optimize based on signal intensity)

  • Wash thoroughly with PBS containing 0.1% Tween-20

  • Counterstain nuclei with DAPI if desired

  • Mount and visualize using appropriate filters for FITC detection (excitation ~495 nm, emission ~519 nm)

• Photobleaching considerations: Minimize exposure to light during all steps and storage to prevent photobleaching of the FITC fluorophore.

• Autofluorescence management: Include controls to account for tissue autofluorescence, particularly in tissues with high intrinsic fluorescence like retina.

• Validated cell types: A549 cells have been positively tested for SFRP5 detection using immunofluorescence approaches .

What are the optimal conditions for immunohistochemical detection of SFRP5?

For successful immunohistochemical detection of SFRP5 in tissue samples:

• Tissue preparation: Formalin-fixed, paraffin-embedded tissues have been successfully used for SFRP5 detection. Human colon tissue has been validated at 5 μg/ml antibody concentration .

• Antigen retrieval: Two validated options exist:

  • TE buffer pH 9.0 (primary recommendation)

  • Citrate buffer pH 6.0 (alternative method)
    The choice depends on tissue type and fixation conditions.

• Antibody dilution: Use at 1:50-1:500 dilution range, with optimization recommended for each tissue type .

• Validated tissues: Positive IHC detection has been confirmed in:

  • Human tissues: thyroid cancer tissue, cervical cancer tissue, colon tissue

  • Animal tissues: mouse heart tissue, rat eye tissue

• Detection system: Standard HRP-DAB detection systems are compatible with SFRP5 antibody detection.

How should unusual SFRP5 expression patterns or band sizes be interpreted?

When encountering unexpected results with SFRP5 antibody:

• Multiple bands or unexpected molecular weights: While the predicted molecular weight of SFRP5 is 36 kDa, observed weights between 36-42 kDa are common . This variation may result from:

  • Post-translational modifications including glycosylation

  • Tissue-specific isoforms

  • Protein degradation products

  • Cross-reactivity with related SFRP family members

• Tissue-specific expression patterns: Expression levels vary significantly between tissues, with highest expression in retinal pigment epithelium and moderate expression in pancreas . Unexpected absence in these tissues may indicate technical issues, while unexpected presence in other tissues may represent pathological conditions or previously undocumented expression patterns.

• Species differences: Human, mouse, and rat SFRP5 share high homology but may show species-specific banding patterns or expression levels. Consider using species-specific positive controls.

• Data validation approaches:

  • Confirm with alternative SFRP5 antibodies targeting different epitopes

  • Validate with mRNA expression data (RT-PCR or RNA-seq)

  • Use knockout/knockdown controls where available

  • Compare with published literature on tissue-specific expression patterns

What are common sources of background or non-specific binding with SFRP5 antibodies and how can they be minimized?

When troubleshooting high background or non-specific binding:

• Antibody-specific considerations:

  • Polyclonal antibodies (like most available SFRP5 antibodies) may exhibit more background than monoclonals

  • For FITC-conjugated antibodies, photobleaching can reduce specific signal relative to background

  • Storage conditions (-20°C or -80°C recommended) significantly impact antibody performance

• Application-specific optimization strategies:

  • Western blot: Increase blocking time/concentration, optimize antibody dilution (1:1000 may reduce background compared to 1:500), increase wash duration/frequency

  • Immunofluorescence: Include autofluorescence controls, optimize fixation to reduce autofluorescence, use Sudan Black B to reduce tissue autofluorescence

  • Immunohistochemistry: Optimize antigen retrieval conditions, use more stringent washing, ensure complete blocking of endogenous peroxidases

• Sample-specific considerations:

  • Adipose tissue may require additional blocking steps due to high lipid content

  • Retinal tissue has high intrinsic autofluorescence requiring specific countermeasures

  • Fixed tissues may require extended blocking to reduce non-specific binding

• Use of appropriate controls:

  • Include secondary-only controls (for non-conjugated antibodies)

  • Include isotype controls to identify non-specific binding

  • Use tissue-specific negative controls where SFRP5 expression is minimal or absent

How can SFRP5 antibodies be implemented in multi-color immunofluorescence studies?

For complex multi-color immunofluorescence studies incorporating SFRP5 detection:

• Fluorophore selection and spectral compatibility: FITC-conjugated SFRP5 antibody (excitation ~495 nm, emission ~519 nm) can be combined with:

  • Red fluorophores (e.g., Texas Red, Cy3) for dual labeling

  • Far-red fluorophores (e.g., Cy5, Alexa Fluor 647) for triple labeling

  • DAPI nuclear counterstain (blue)

  Ensure adequate spectral separation between fluorophores and use appropriate filter sets to minimize bleed-through.

• Co-localization studies: SFRP5 can be co-localized with:

  • Wnt pathway components to study interaction dynamics

  • Cell type-specific markers in retina (e.g., rhodopsin for photoreceptors)

  • Adipocyte markers in metabolic studies

  • Inflammatory markers in obesity/diabetes models

• Sequential immunostaining protocol:

  • Begin with the least abundant target (often SFRP5)

  • Use carefully titrated antibody dilutions to prevent overcrowding of epitopes

  • Consider implementing tyramide signal amplification for low-abundance targets

  • Use nuclear counterstaining as the final step

• Image acquisition and analysis considerations:

  • Capture single-channel images sequentially to minimize spectral overlap

  • Implement appropriate background subtraction for each channel

  • Use co-localization analysis software with appropriate statistical validation

  • Consider super-resolution microscopy techniques for detailed co-localization analysis

What are the methodological considerations for studying SFRP5 in metabolic disease models?

For investigating SFRP5's role in metabolic disorders:

• Tissue selection and processing:

  • Adipose tissue (primary site of SFRP5 expression in metabolic context)

  • Liver (for metabolic disease complications)

  • Pancreas (where SFRP5 shows moderate expression)

  • Blood samples (for circulating SFRP5 levels)

• Experimental design considerations:

  • Compare SFRP5 expression between healthy, pre-diabetic, and diabetic models

  • Analyze expression changes in response to metabolic interventions (diet, exercise, pharmaceuticals)

  • Consider time-course studies to track expression during disease progression

  • Implement tissue-specific knockout models to establish causality

• Complementary analytical approaches:

  • Combine immunodetection with ELISA quantification (Human sFRP-5 DuoSet ELISA)

  • Correlate protein expression with functional metabolic readouts

  • Integrate with transcriptomic and proteomic analyses

  • Assess relationship between SFRP5 expression and inflammatory markers

• Technical validation approaches:

  • Use multiple antibodies targeting different epitopes

  • Validate protein expression changes with mRNA quantification

  • Compare results across multiple species/models

  • Implement appropriate statistical analyses for metabolic data correlation

How can SFRP5 antibodies be utilized in studies of retinal development and pathology?

For retinal research applications:

• Developmental studies protocol:

  • Use timed samples across developmental stages

  • Compare SFRP5 expression patterns with established developmental markers

  • Implement both sectioned tissues and whole-mount preparations

  • Consider conditional knockout approaches to study temporal requirements

• Cellular localization in retinal architecture:

  • SFRP5 is highly expressed in retinal pigment epithelium

  • May be involved in photoreceptor polarity determination

  • Combine with layer-specific markers for precise localization

  • Use confocal microscopy for accurate spatial resolution

• Pathological contexts to consider:

  • Retinal degeneration models

  • Polarity defects in photoreceptors

  • Age-related macular degeneration

  • Diabetic retinopathy (connecting to SFRP5's metabolic functions)

• Species considerations:

  • Mouse retina tissue has confirmed SFRP5 expression

  • Rat eye tissue shows positive immunohistochemical detection

  • Consider species-specific differences in retinal architecture when interpreting results

How can SFRP5 antibodies be incorporated into high-throughput screening approaches?

For implementing SFRP5 detection in high-throughput contexts:

• Automated immunohistochemistry/immunofluorescence platforms:

  • Optimize antibody concentration for automated systems

  • Develop standardized positive controls for quality assurance

  • Establish clear threshold criteria for SFRP5 positivity

  • Implement image analysis algorithms for quantitative assessment

• Multiplex approaches:

  • Combine SFRP5 detection with other Wnt pathway components

  • Develop multiplex ELISA systems incorporating SFRP5

  • Consider multiplex approaches combining protein and transcript detection

  • Implement tissue microarray approaches for high-throughput screening

• Screening applications:

  • Drug discovery platforms targeting Wnt modulation

  • Compound screening for metabolic disease intervention

  • Genetic screening approaches correlating SFRP5 expression with phenotypes

  • Patient stratification based on SFRP5 expression patterns

• Quantitative considerations:

  • Develop robust quantification approaches for SFRP5 signal intensity

  • Establish normalization strategies against housekeeping proteins

  • Implement quality control measures specific to fluorescent applications

  • Consider machine learning approaches for complex pattern recognition

What are the technical considerations for studying SFRP5 protein-protein interactions?

For investigating SFRP5's interactions with binding partners:

• Co-immunoprecipitation approaches:

  • Use SFRP5 antibody for pulldown experiments followed by detection of Wnt proteins

  • Alternatively, use tagged Wnt proteins to pull down SFRP5

  • Consider crosslinking approaches for transient interactions

  • Implement stringent washing conditions to eliminate non-specific binding

• Proximity ligation assays:

  • Combine SFRP5 antibody with antibodies against potential binding partners

  • Look for signal amplification indicating proximity within 40 nm

  • Quantify interaction frequency in different tissues/conditions

  • Compare with co-localization results from standard immunofluorescence

• Microscopy-based interaction studies:

  • Implement FRET (Fluorescence Resonance Energy Transfer) approaches using appropriate fluorophore pairs

  • Consider FLIM (Fluorescence Lifetime Imaging Microscopy) for more precise interaction detection

  • Use super-resolution microscopy to resolve spatial relationships at nanometer scale

  • Combine with live-cell imaging where possible to track dynamic interactions

• Validation strategies:

  • Confirm interactions using multiple methodological approaches

  • Utilize domain deletion mutants to map interaction regions

  • Implement competitive binding approaches with recombinant proteins

  • Correlate protein interaction data with functional outcomes