FGF16 Antibody, HRP conjugated

What is FGF16 and what are its primary biological functions?

FGF16 (Fibroblast Growth Factor 16) belongs to the heparin-binding growth factors family. It functions primarily as a secreted protein that induces hepatocellular proliferation . Recent studies have demonstrated that FGF16 plays significant roles in multiple cancers, including embryonic carcinoma, ovarian cancer, and liver cancer . Notably, FGF16 has been found to be overexpressed in lung cancer tissues, where it contributes to cell proliferation . Research in rat models has also shown that FGF16 can stimulate proliferation while blocking differentiation in Leydig cells, indicating its complex role in cellular development .

What are the key specifications of FGF16 Antibody, HRP conjugated?

FGF16 Antibody, HRP conjugated is a polyclonal antibody raised in rabbits using KLH-conjugated synthetic peptides derived from human FGF16 (immunogen range: 121-207/207) . The antibody is conjugated to Horseradish Peroxidase (HRP), which facilitates direct detection without the need for secondary antibodies in various applications. It has a concentration of 1μg/μl and is stored in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol . The antibody demonstrates cross-reactivity with multiple species, including human, mouse, rat, cow, sheep, pig, horse, and chicken samples .

What are the recommended applications for FGF16 Antibody, HRP conjugated?

The FGF16 Antibody, HRP conjugated has been validated for multiple experimental applications, including:

These applications provide researchers with versatile options for detecting FGF16 in various experimental contexts, from protein expression analysis to tissue localization studies .

How should I design Western blot experiments using FGF16 Antibody, HRP conjugated?

When designing Western blot experiments with FGF16 Antibody, HRP conjugated, start with protein extraction using RIPA buffer for cell lysis and protein isolation . For FGF16 detection in lung cancer cells, researchers have successfully used SDS-PAGE followed by transfer to PVDF membranes (Millipore) . Block membranes with 5% skim milk solution in PBS before antibody application .

For optimal results, use the antibody at dilutions between 1:300-5000 depending on your protein concentration and detection system sensitivity . Since this antibody is HRP-conjugated, you can directly visualize results using ECL (Enhanced Chemiluminescence) without requiring a secondary antibody step . This streamlines the protocol and reduces potential background. Include appropriate loading controls (e.g., β-actin) and positive controls (tissues known to express FGF16) for result validation .

What is the optimal protocol for immunohistochemistry with FGF16 Antibody, HRP conjugated?

For immunohistochemistry using FGF16 Antibody, HRP conjugated, the protocol differs slightly between paraffin-embedded (IHC-P) and frozen (IHC-F) sections:

For paraffin-embedded tissues:

• Deparaffinize sections and perform antigen retrieval (citrate buffer pH 6.0 is commonly effective)

• Block endogenous peroxidase with 3% hydrogen peroxide

• Block non-specific binding with 5% normal serum

• Apply FGF16 Antibody, HRP conjugated at 1:200-400 dilution and incubate overnight at 4°C

• Rinse thoroughly with PBS

• Develop with DAB substrate

• Counterstain, dehydrate, and mount

For frozen sections:

• Fix sections briefly with acetone or 4% paraformaldehyde

• Block endogenous peroxidase activity

• Apply FGF16 Antibody, HRP conjugated at a higher concentration (1:100-500) than for paraffin sections

• Proceed with development and counterstaining as above

Research examining FGF16 expression in clinical samples has demonstrated the utility of immunohistochemistry for visualizing FGF16 distribution and expression levels in tissues like lung cancer where FGF16 is overexpressed compared to normal tissues .

How can I determine if FGF16 is a direct target of specific miRNAs using FGF16 Antibody?

To investigate whether FGF16 is regulated by specific miRNAs (such as miR-520b in lung cancer), implement a multi-step experimental approach:

• Prediction and Cloning: Use bioinformatics tools (e.g., TargetScan) to predict miRNA binding sites on FGF16 mRNA. Clone the wild-type and mutant 3′UTR fragments containing the predicted binding sites into a luciferase reporter vector (e.g., pGL3-control vector) .

• Transfection Studies: Transfect cells with the miRNA of interest at different concentrations along with the reporter constructs. Include appropriate controls (empty vector, non-targeting miRNA) .

• Luciferase Assay: Measure luciferase activity to determine if the miRNA directly interacts with the FGF16 3′UTR. A decrease in luciferase activity with increasing miRNA concentration indicates direct binding .

• Protein Expression Validation: Use FGF16 Antibody, HRP conjugated (1:300-5000 dilution) for Western blotting to confirm that miRNA transfection reduces FGF16 protein levels .

• Functional Validation: Perform functional assays (e.g., proliferation, migration) with miRNA mimics or inhibitors, and rescue experiments by overexpressing FGF16 .

This comprehensive approach has been validated in research demonstrating that miR-520b directly targets FGF16 in lung cancer, causing mRNA cleavage and reduced protein expression .

What methods can I use to study the role of FGF16 in cell proliferation using this antibody?

To investigate FGF16's role in cell proliferation, several methodological approaches can be employed:

• Cell Proliferation Assays:

  • MTT assay: Assess cellular metabolic activity as a proxy for proliferation. Studies in lung cancer cells have shown that modulating FGF16 levels (through miR-520b or siRNA targeting FGF16) significantly affects proliferation rates .

  • EdU incorporation assay: Directly measure DNA synthesis during cell proliferation. This has been successfully used to demonstrate FGF16's effect on stem Leydig cell proliferation in vitro .

  • PCNA immunostaining: Use anti-PCNA antibodies alongside FGF16 Antibody to correlate FGF16 expression with proliferating cell nuclear antigen positivity .

• Knockdown and Overexpression Studies:

  • siRNA-mediated FGF16 knockdown: Transfect cells with FGF16-specific siRNAs and confirm knockdown efficiency via Western blotting using FGF16 Antibody, HRP conjugated .

  • FGF16 overexpression: Clone FGF16 cDNA into an expression vector and transfect cells, then confirm expression using the antibody.

• Pathway Analysis:

  • Western blotting for downstream signaling proteins (e.g., phosphorylated AKT1/2, ERK1/2) to determine potential mechanisms of FGF16-induced proliferation .

These approaches have been validated in studies showing that FGF16 increases phosphorylation of AKT1/2 and ERK1/2 in vivo, suggesting these pathways mediate its proliferative effects .

How can I resolve conflicting results between FGF16's reported roles in different tissue contexts?

Resolving conflicting results regarding FGF16's roles across different tissues requires a systematic approach:

• Tissue-Specific Expression Analysis:

  • Perform comparative immunohistochemistry (IHC-P at 1:200-400 dilution) across multiple tissue types using FGF16 Antibody, HRP conjugated .

  • Quantify expression levels in different tissues and cell types to establish tissue-specific baselines.

• Context-Dependent Signaling Assessment:

  • Investigate tissue-specific co-factors that might modulate FGF16 activity. For example, FGF16 increases Leydig cell proliferation while blocking differentiation in testicular tissue , but promotes both proliferation and cancer progression in lung tissue .

  • Examine pathway crosstalk by analyzing multiple signaling pathways simultaneously (AKT, ERK, others specific to the tissue context).

• Experimental Models Comparison:

  • Compare results across different experimental models (cell lines vs. primary cells vs. in vivo models).

  • Use the same antibody concentrations and detection methods across experiments for valid comparisons.

  • Consider species differences, as the antibody shows cross-reactivity with multiple species .

• Dose-Response Relationships:

  • Conduct dose-response studies (as performed with 0, 10, and 100 ng/testis FGF16 in rat testis) to identify potential concentration-dependent effects that might explain different outcomes.

By methodically addressing these factors, researchers can resolve apparent contradictions, such as why FGF16 stimulates proliferation in certain contexts while having distinct effects on differentiation or other cellular processes in others.

What approaches can help differentiate between direct and indirect effects of FGF16 on gene expression?

Differentiating between direct and indirect effects of FGF16 on gene expression requires multiple complementary approaches:

• Temporal Analysis:

  • Perform time-course experiments treating cells with recombinant FGF16 and collecting samples at multiple time points (15min, 30min, 1h, 2h, 6h, 24h).

  • Use FGF16 Antibody, HRP conjugated in Western blotting (1:300-5000 dilution) to confirm stable FGF16 levels throughout the experiment .

  • Analyze expression of potential target genes (e.g., Leydig cell genes like Lhcgr, Scarb1, Star) .

  • Early-response genes are more likely to be direct targets.

• Pathway Inhibition Studies:

  • Pretreat cells with inhibitors of known FGF16 downstream pathways (e.g., PI3K/AKT inhibitors, MAPK/ERK inhibitors) before FGF16 stimulation .

  • If a gene's expression change is blocked by a specific inhibitor, it suggests that pathway mediates the effect.

• Chromatin Immunoprecipitation (ChIP) Analysis:

  • Perform ChIP to identify transcription factors activated by FGF16 signaling.

  • Compare these binding sites with promoters of differentially expressed genes.

• Integration of Multiple Data Types:

  • Combine RNA-seq/qPCR data with protein expression data (using FGF16 Antibody, HRP conjugated in Western blots) to identify discrepancies that might indicate post-transcriptional regulation.

  • Incorporate data from studies showing FGF16's effects on gene expression in different contexts, such as downregulation of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1, Hsd17b3) and Sertoli cell genes (Fshr, Dhh, Sox9) .

These approaches provide a comprehensive framework for discriminating between genes directly regulated by FGF16 signaling versus those affected through secondary mechanisms.

What are the most common issues when using FGF16 Antibody, HRP conjugated, and how can they be resolved?

When working with FGF16 Antibody, HRP conjugated, researchers may encounter several common challenges:

• High Background in Western Blots:

  • Problem: Non-specific binding resulting in high background signal.

  • Solution: Optimize blocking (increase blocking time to 2 hours with 5% skim milk) , use higher dilution of antibody (start at 1:5000 and adjust as needed) , and increase washing steps (4-5 washes of 10 minutes each with TBST).

• Weak or No Signal:

  • Problem: Insufficient antibody binding or low FGF16 expression.

  • Solution: Decrease antibody dilution (try 1:300-1:1000) , increase protein loading, optimize antigen retrieval methods, or verify FGF16 expression in your sample type before proceeding.

• Variable Results Between Experiments:

  • Problem: Inconsistent antibody performance across experiments.

  • Solution: Aliquot antibody upon receipt to avoid repeated freeze-thaw cycles as recommended in storage conditions , standardize protein extraction methods, and include positive controls (tissues known to express FGF16, such as lung cancer samples) .

• Cross-Reactivity Issues:

  • Problem: Non-specific binding to proteins other than FGF16.

  • Solution: Validate specificity using FGF16 knockdown controls, test multiple dilutions to find optimal signal-to-noise ratio, and consider blocking with additional BSA (2-5%).

• IHC Optimization Challenges:

  • Problem: Poor staining quality in immunohistochemistry.

  • Solution: Optimize antigen retrieval methods, adjust antibody concentration between the recommended ranges (1:200-400 for IHC-P, 1:100-500 for IHC-F) , and extend incubation times (overnight at 4°C may improve signal).

Implementing these troubleshooting approaches will help ensure reliable and reproducible results when using FGF16 Antibody, HRP conjugated.

How can I validate the specificity of FGF16 Antibody, HRP conjugated in my experimental system?

Validating antibody specificity is crucial for reliable experimental results. For FGF16 Antibody, HRP conjugated, implement these validation strategies:

• Positive and Negative Controls:

  • Positive Controls: Include samples known to express FGF16, such as lung cancer tissues where FGF16 is overexpressed .

  • Negative Controls: Use tissues or cells with low/no FGF16 expression or samples where FGF16 has been knocked down via siRNA .

• RNAi-Mediated Knockdown:

  • Transfect cells with FGF16-specific siRNA and control siRNA.

  • Perform Western blotting using FGF16 Antibody, HRP conjugated (1:300-5000 dilution) .

  • A specific antibody will show significantly reduced signal in knockdown samples compared to controls.

• Molecular Weight Verification:

  • Confirm that the detected band appears at the expected molecular weight for FGF16.

  • Include molecular weight markers to accurately assess protein size.

• Peptide Competition Assay:

  • Pre-incubate the antibody with the immunizing peptide (derived from human FGF16, range 121-207/207) .

  • A specific antibody's signal should be significantly reduced or eliminated after peptide competition.

• Cross-Species Reactivity Testing:

  • If working with non-human samples, validate the antibody's performance in your species of interest, even though the antibody is predicted to react with multiple species (human, mouse, rat, cow, sheep, pig, horse, chicken) .

• Multiple Detection Methods:

  • Compare results from different applications (WB, ELISA, IHC-P, IHC-F) to ensure consistent detection patterns.

  • Discrepancies across methods might indicate specificity issues that need to be addressed.

Thorough validation ensures that experimental findings truly reflect FGF16 biology rather than artifacts of non-specific antibody binding.