FRZB Antibody

What is the optimal protocol for FRZB detection in Western Blot applications?

For optimal FRZB detection via Western Blot, the recommended dilution ranges from 1:5000 to 1:50000, though this should be titrated for each specific testing system . Standard protocols involve:

• Sample preparation with proper lysis buffers containing protease inhibitors

• Protein separation on SDS-PAGE (typically detecting FRZB at approximately 36 kDa)

• Transfer to PVDF or nitrocellulose membrane

• Blocking with appropriate buffer (typically 5% non-fat milk or BSA)

• Primary antibody incubation (overnight at 4°C recommended)

• Secondary antibody incubation

• Signal detection via chemiluminescence

When troubleshooting, consider that FRZB is a secreted protein, so cell culture media may need to be concentrated for optimal detection .

Which tissue types demonstrate significant FRZB expression?

FRZB expression varies significantly across tissue types. Research indicates that:

• Normal human primary cell lines show higher baseline FRZB mRNA levels compared to soft tissue sarcoma (STS) cell lines

• Skeletal muscle demonstrates increased FRZB expression in denervation conditions such as ALS

• Endomysial connective tissue shows predominant FRZB localization with some expression at muscle membranes

• Joint tissues in developmental dysplasia of the hip (DDH) exhibit elevated FRZB expression

When designing experiments, consider that FRZB expression can be significantly altered in pathological conditions compared to normal tissues.

What are the recommended immunofluorescence protocols for FRZB detection in tissue sections?

For immunofluorescence detection of FRZB in tissue sections:

• Fix sections in 4% paraformaldehyde

• Wash with TBS-T buffer

• Perform antigen retrieval (for paraffin sections, use 1mM EDTA at 80°C for 15 minutes)

• Block with goat serum to minimize non-specific binding

• Incubate with anti-FRZB primary antibody at 1:200 dilution for 2 hours at room temperature

• Detect using fluorescent-conjugated secondary antibodies (e.g., Alexa Fluor 555)

• Counterstain nuclei with DAPI

For optimal results, consider hyaluronidase treatment before antibody incubation when working with cartilaginous or joint tissues.

How can FRZB antibodies be utilized to study the interplay between Wnt signaling and c-Met pathways in sarcomas?

FRZB antibodies provide valuable tools for investigating the complex relationship between Wnt signaling and c-Met pathways in sarcomas:

• Implement co-immunoprecipitation with FRZB antibodies to identify binding partners in the signaling cascades

• Perform immunoblotting for both FRZB and c-Met in parallel to demonstrate inverse correlation patterns

• Utilize FRZB antibodies in chromatin immunoprecipitation (ChIP) assays to identify transcriptional targets

• Conduct immunohistochemistry on tissue microarrays to compare FRZB and c-Met expression patterns

Research has demonstrated a significant inverse correlation between FRZB and Met expression in both STS cell lines and tissues (Spearman rank order correlation coefficient: –0.47; P < 0.05) . This relationship suggests FRZB as a potential therapeutic target for modulating Met signaling in STS treatment.

What methodological approaches can resolve contradictory data regarding FRZB's role as tumor suppressor versus oncogene?

To address contradictory findings regarding FRZB's dual roles:

• Conduct context-specific expression analysis using FRZB antibodies across different tumor types

• Implement tissue-specific knockdown/overexpression models followed by antibody validation

• Perform domain-specific antibody detection to identify potential isoform differences

• Analyze post-translational modifications using phospho-specific FRZB antibodies

Research indicates that FRZB acts as a tumor suppressor in soft tissue sarcomas and prostate cancer , while potentially functioning as an oncogene in metastatic renal cancer . These context-dependent functions likely relate to tissue-specific Wnt signaling networks and interaction partners.

What experimental design best elucidates FRZB's role in epithelial-mesenchymal transition?

To investigate FRZB's involvement in epithelial-mesenchymal transition (EMT):

• Implement FRZB knockdown/overexpression in relevant cell models

• Monitor changes in epithelial markers (keratins 8 and 18) and mesenchymal markers (vimentin, N-cadherin, fibronectin, Slug, and Twist) via immunoblotting

• Confirm protein localization changes using immunofluorescence with FRZB antibodies

• Assess functional changes in invasion and motility following FRZB modulation

Studies show that FRZB transfection in fibrosarcoma (HT1080) and liposarcoma (SW872) cells significantly reduced cellular invasion, motility, and colony formation in soft agar . These effects were associated with up-regulation of epithelial markers and down-regulation of mesenchymal markers, suggesting FRZB may reverse or inhibit EMT processes in certain contexts.

How should researchers address cross-reactivity concerns when using FRZB antibodies?

To minimize cross-reactivity issues:

• Validate antibody specificity using positive and negative control samples

• Implement FRZB knockdown/knockout controls to confirm specificity

• Consider using multiple antibodies targeting different FRZB epitopes

• Perform pre-adsorption tests with recombinant FRZB protein

Available FRZB antibodies have demonstrated reactivity with human and mouse samples , but thorough validation is essential, particularly when exploring species with less characterized FRZB homology.

What are the optimal approaches for quantifying FRZB expression in clinical samples?

For accurate FRZB quantification in clinical samples:

• Implement standardized immunohistochemistry protocols with tissue microarrays

• Develop scoring systems based on staining intensity and percentage of positive cells

• Utilize digital pathology algorithms for unbiased quantification

• Consider multiplex staining to correlate FRZB expression with other biomarkers

A comprehensive approach involves correlating protein-level detection with mRNA quantification via qRT-PCR, as demonstrated in studies comparing FRZB expression between normal and STS cell lines .

How can FRZB antibodies facilitate the investigation of FRZB's role in chondrogenesis and joint development?

To explore FRZB's functions in chondrogenesis:

• Implement FRZB immunoprecipitation followed by mass spectrometry to identify novel binding partners

• Conduct immunofluorescence co-localization studies during chondrogenic differentiation

• Perform chromatin immunoprecipitation with FRZB antibodies to identify downstream transcriptional targets

• Analyze FRZB expression patterns in developmental time-course experiments

Research has demonstrated that FRZB knockdown significantly decreases chondrogenesis in ATDC5 cells, affecting genes involved in cartilage development including ACAN, SOX9, and DKK1 . These findings suggest FRZB's crucial role in joint development, with potential implications for conditions like developmental dysplasia of the hip.

What methodological approaches can elucidate FRZB's involvement in neuromuscular diseases?

To investigate FRZB in neuromuscular diseases:

• Implement longitudinal immunohistochemical analyses of FRZB expression in disease models

• Correlate FRZB levels with denervation markers using multiplex immunofluorescence

• Perform subcellular fractionation followed by immunoblotting to track FRZB localization

• Conduct co-immunoprecipitation assays to identify disease-specific interaction partners

Studies have shown that FRZB is upregulated in the early stages of disease in the SOD1^G93A mouse model of ALS, with increased immunoreactivity surrounding atrophied myofibers . This suggests FRZB as a potential biomarker for muscle denervation with relevance to disease progression monitoring.

How can researchers optimize antibody-based detection of FRZB in the context of signaling pathway analysis?

For comprehensive signaling pathway analysis:

• Implement proximity ligation assays to detect FRZB-protein interactions in situ

• Use phospho-specific antibodies to detect downstream effectors following FRZB modulation

• Conduct time-course experiments with synchronized intervention and antibody detection

• Perform competitive binding assays between FRZB and Wnt ligands

Research has demonstrated that FRZB reduces LEF/TCF transcriptional activity by 68-73% in sarcoma cell lines, with similar effects observed with dominant-negative LRP5 receptor . When investigating Wnt signaling, assessment of β-catenin levels and localization provides important complementary data to FRZB detection.

How should researchers interpret discrepancies between FRZB mRNA and protein levels in experimental settings?

To address mRNA-protein level discrepancies:

• Implement parallel qRT-PCR and immunoblotting in time-course experiments

• Consider post-transcriptional regulation by analyzing miRNA levels that target FRZB

• Assess protein stability through cycloheximide chase experiments

• Evaluate secretion efficiency by comparing intracellular versus secreted FRZB levels

Post-transcriptional regulation may significantly impact FRZB expression, as suggested by studies investigating potential upstream miRNAs for FRZB in developmental dysplasia of the hip . When analyzing experimental data, consider the entire regulatory landscape from transcription to secretion.

What controls are essential for validating FRZB antibody specificity in functional studies?

Essential controls include:

• FRZB knockdown/knockout samples to confirm signal specificity

• Recombinant FRZB protein as positive control (50 ng/ml has been used in functional studies)

• FRZB neutralizing antibody (200 ng/ml) to confirm functional blockade

• Isotype controls for immunoprecipitation and immunofluorescence applications

In functional studies, FRZB siRNA has been successfully employed to validate antibody specificity and to investigate FRZB's role in chondrogenesis, with sequences targeting specific regions of FRZB mRNA .