FNDC3B Antibody

What is FNDC3B and why is it significant in research?

FNDC3B, also known as factor for adipocyte differentiation 104 (FAD104) or HCV NS5A-binding protein 37, is a 1,204 amino acid protein characterized by nine fibronectin type-III domains . It plays a critical role in several biological processes including:

• Adipogenesis: FNDC3B is expressed during early adipogenesis and functions as a positive regulator of this process

• Cell adhesion and migration: Studies with FNDC3B-deficient mouse embryonic fibroblasts demonstrate impaired stress fiber formation, suggesting its involvement in cell adhesion, spreading, and migration

• Development: FNDC3B-deficient mice exhibit a lethal phenotype within one day of birth, indicating its essential role in survival and development

• Cancer progression: FNDC3B exhibits oncogenic characteristics in various tumor types, including glioma, pancreatic cancer, and cervical cancer

The multifunctional nature of FNDC3B makes antibodies against this protein valuable tools for investigating diverse physiological and pathological processes.

FNDC3B primarily localizes to the membrane and endoplasmic reticulum . According to The Human Protein Atlas, immunofluorescence staining with anti-FNDC3B antibodies reveals specific subcellular patterns that can be visualized using appropriate markers .

When performing immunofluorescence experiments, researchers should expect to observe:

• Membrane staining (FNDC3B is characterized as a single-pass membrane protein)

• Potential endoplasmic reticulum localization

• Specific staining patterns in certain cell types, such as epithelial cells, adipocytes, and cells of neuronal origin

For optimal visualization, use confocal microscopy with appropriate co-staining for cellular compartments to precisely determine FNDC3B localization in your specific cell type of interest.

How can FNDC3B antibodies be used to investigate immune infiltration in cancer microenvironments?

FNDC3B has emerged as a promising immunotherapeutic biomarker, particularly in glioma . Research indicates that FNDC3B expression correlates with immune cell infiltration patterns and immune checkpoint molecules . Methodological approaches using FNDC3B antibodies include:

• Correlation analyses between FNDC3B expression and immune cell infiltration:

  • Research has shown that FNDC3B expression positively correlates with the abundance of various immune cells including central memory CD8 T cells (r = 0.497), effector memory CD8 T cells (r = 0.412), regulatory T cells (r = 0.521), NK cells (r = 0.532), NKT cells (r = 0.64), memory B cells (r = 0.64), and macrophages (r = 0.349)

• Investigation of FNDC3B association with immune checkpoint molecules:

  • Significant positive correlations have been observed between FNDC3B expression and immune checkpoint molecules: B7-H3 (R = 0.69), PD-L1 (R = 0.58), TIM-3 (R = 0.43), PD-1 (R = 0.42), CTLA-4 (R = 0.34), and OX40 (R = 0.31)

• Profiling immune infiltration based on FNDC3B expression levels:

  • Using techniques like immunohistochemistry with FNDC3B antibodies combined with immune cell markers

  • Correlating FNDC3B expression with immune, stromal, and ESTIMATE scores

  • Analyzing the proportions of 22 types of immune cells using CIBERSORTx in FNDC3B high vs. low expression groups

These approaches can provide valuable insights into the role of FNDC3B in modulating the tumor immune microenvironment and potentially identify novel immunotherapeutic targets.

What is the significance of FNDC3B as a prognostic biomarker and how can antibodies help establish this role?

FNDC3B has been identified as a potential prognostic biomarker in several cancer types, including glioma and pancreatic cancer . Researchers can utilize FNDC3B antibodies to:

By combining antibody-based detection methods with statistical analysis of clinical data, researchers can establish the value of FNDC3B as a prognostic biomarker in various cancer types.

How can FNDC3B antibodies be employed to study its role in adipogenesis and osteoblast differentiation?

FNDC3B has been reported to regulate adipogenesis and osteoblast differentiation . Researchers can use FNDC3B antibodies to investigate these processes through:

• Temporal expression analysis during differentiation:

  • Track FNDC3B protein expression at different stages of adipocyte or osteoblast differentiation using western blotting

  • Perform immunofluorescence to visualize changes in FNDC3B localization during differentiation

• Functional studies with knockdown/overexpression:

  • Use FNDC3B antibodies to confirm successful knockdown or overexpression in experimental models

  • Correlate FNDC3B expression levels with differentiation markers

• Co-immunoprecipitation to identify interaction partners:

  • Use FNDC3B antibodies for co-IP experiments to identify protein-protein interactions that mediate its effects on differentiation

  • Combine with mass spectrometry to identify novel interaction partners

• Chromatin immunoprecipitation (ChIP) studies:

  • If FNDC3B functions as a transcriptional regulator, ChIP using FNDC3B antibodies can identify its genomic targets during differentiation

These experimental approaches can provide mechanistic insights into how FNDC3B regulates cell fate decisions in mesenchymal lineages.

Experimental Design and Optimization

Ensuring antibody specificity is crucial for generating reliable data. Researchers should implement the following validation strategies:

• Multiple antibody approach:

  • Use antibodies from different suppliers or those recognizing different epitopes

  • Compare staining/detection patterns for consistency

• Genetic approaches:

  • Use FNDC3B knockout/knockdown cells as negative controls

  • Use FNDC3B overexpression systems as positive controls

  • Verify that signal intensity correlates with expression level

• Blocking peptide validation:

  • Pre-incubate antibody with the immunizing peptide

  • Compare signal with and without peptide blockade; specific signals should be eliminated

• Cross-reactivity assessment:

  • Test antibody on samples from multiple species if conducting comparative studies

  • Verify specificity for FNDC3B versus other FNDC family members

• Orthogonal validation:

  • Confirm protein expression using independent methods (e.g., mass spectrometry)

  • Correlate protein detection with mRNA expression data

These validation approaches will ensure that experimental findings truly reflect FNDC3B biology rather than non-specific antibody interactions.

What are the key considerations for selecting the appropriate FNDC3B antibody for specific research questions?

When selecting an FNDC3B antibody for your research, consider:

• Epitope location:

  • Different antibodies recognize distinct regions of FNDC3B (e.g., AA 5-71, AA 176-205, AA 250-299, AA 921-1020)

  • For studying protein domains, select antibodies targeting the region of interest

  • For detecting potential splice variants or processed forms, consider epitope location relative to these features

• Species reactivity:

  • Ensure the antibody recognizes FNDC3B in your experimental species

  • Available antibodies have varying cross-reactivity profiles (human-only vs. human/mouse/rat reactivity)

• Application compatibility:

  • Verify that the antibody has been validated for your specific application

  • Some antibodies perform well in multiple applications while others are application-specific

• Clonality considerations:

  • Monoclonal antibodies offer high specificity for a single epitope and batch-to-batch consistency

  • Polyclonal antibodies recognize multiple epitopes, potentially providing stronger signals but with possible increased background

• Form and conjugation:

  • For multiplexing experiments, consider directly conjugated antibodies

  • For signal amplification, unconjugated primary antibodies with secondary detection may be preferable

• Validation evidence:

  • Review available validation data including western blots, immunohistochemistry images, etc.

  • Consider antibodies with validation in experimental systems similar to yours

Carefully matching antibody properties to your experimental needs will maximize the likelihood of successful results.

How can researchers address common issues with FNDC3B antibody staining in immunohistochemistry?

Several challenges may arise when using FNDC3B antibodies for immunohistochemistry. Here are solutions to common problems:

• Weak or absent staining:

  • Optimize antigen retrieval: FNDC3B is a membrane-associated protein that may require aggressive antigen retrieval; try citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Increase antibody concentration: If using a 1:100 dilution, try 1:50

  • Extend primary antibody incubation: Consider overnight incubation at 4°C

  • Use signal amplification systems: Try biotin-streptavidin or polymer-based detection systems

• High background staining:

  • Improve blocking: Use 5-10% normal serum from the species of the secondary antibody

  • Reduce antibody concentration: If using 1:50, try 1:100 or 1:150

  • Include protein blockers: Add 1% BSA to antibody diluent

  • Ensure proper washing: Extend wash steps with agitation

• Non-specific staining:

  • Validate with positive and negative controls

  • Perform antibody pre-absorption with immunizing peptide

  • Optimize fixation time: Overfixation can create artifacts

  • Use monoclonal antibodies if polyclonal antibodies show non-specific binding

• Inconsistent staining across tissue sections:

  • Standardize tissue processing: Control fixation time and processing conditions

  • Use automated staining platforms if available

  • Prepare larger volumes of antibody dilutions to reduce variability

Always validate staining patterns by comparing with published data on FNDC3B expression and localization.

How should researchers interpret apparent discrepancies in FNDC3B expression data across different detection methods?

Discrepancies in FNDC3B expression data across different methods are common and may reflect biological realities rather than technical issues. Consider these interpretative approaches:

• Understanding methodological differences:

  • Antibody-based methods (IHC, WB, IF) detect protein expression while RT-PCR or RNA-seq measure mRNA levels

  • Discrepancies between mRNA and protein data may reflect post-transcriptional regulation

  • Different antibodies targeting distinct epitopes may give different results if:

    • Protein undergoes post-translational modifications

    • Protein forms complexes that mask epitopes

    • Alternative splicing affects epitope presence

• Resolution strategies:

  • Employ multiple antibodies targeting different regions of FNDC3B

  • Correlate protein detection with mRNA expression data

  • Use orthogonal methods (e.g., mass spectrometry) to confirm expression

• Biological interpretation of discrepancies:

  • Higher mRNA than protein may suggest post-transcriptional regulation

  • Higher protein than mRNA may indicate increased protein stability

  • Differential detection across antibodies may reveal information about protein processing or modification

• Tissue-specific considerations:

  • FNDC3B expression varies across tissues; compare your results with database information

  • Consider the cellular composition of your samples, as FNDC3B expression may vary by cell type

By carefully analyzing discrepancies, researchers can gain deeper insights into FNDC3B biology rather than simply dismissing conflicting data.

What controls are essential when studying FNDC3B in relation to immune infiltration?

When investigating FNDC3B in the context of immune infiltration, proper controls are crucial for accurate interpretation:

• Tissue/cell controls:

  • Include tissues with known high (e.g., glioma, pancreatic cancer) and low FNDC3B expression

  • Use cell lines with established FNDC3B expression levels as benchmarks

  • Include normal adjacent tissue when studying cancer samples to establish baseline expression

• Technical controls for antibody specificity:

  • Include isotype controls to assess non-specific binding

  • Use blocking peptides to confirm signal specificity

  • Include secondary-only controls to detect non-specific secondary antibody binding

• Functional controls when manipulating FNDC3B expression:

  • Include vector-only controls when overexpressing FNDC3B

  • Use non-targeting siRNA/shRNA controls when knocking down FNDC3B

  • Validate expression changes at both mRNA and protein levels

• Controls for immune infiltration analysis:

  • Use established immune cell markers alongside FNDC3B staining

  • Include tissues with known immune infiltration patterns as references

  • Consider multiplexed immunohistochemistry to simultaneously detect FNDC3B and immune cell markers

• Data analysis controls:

  • Analyze correlations between FNDC3B and known immune regulators

  • Compare your findings with published datasets on immune infiltration

  • Include multiple statistical approaches to validate associations

How might FNDC3B antibodies be utilized to explore its potential as an immunotherapeutic target?

FNDC3B's correlation with immune checkpoint molecules and immune infiltration suggests promising applications in cancer immunotherapy research:

• Dual staining approaches:

  • Utilize FNDC3B antibodies alongside immune checkpoint markers (PD-L1, CTLA-4, B7-H3) in patient samples

  • Quantify co-expression patterns to identify patient subgroups that might benefit from combination therapies

  • Perform multiplex immunohistochemistry to visualize the spatial relationship between FNDC3B-expressing cells and tumor-infiltrating lymphocytes

• Functional studies:

  • Use antibodies to confirm FNDC3B knockdown/overexpression in immune modulation studies

  • Investigate how modulating FNDC3B affects response to immune checkpoint blockade in preclinical models

  • Explore the impact of FNDC3B on immune cell recruitment and activation

• Therapeutic antibody development:

  • If FNDC3B is confirmed as a therapeutic target, existing research antibodies can inform the development of therapeutic antibodies

  • Screening antibodies that block functional domains could identify candidates for further development

• Biomarker development:

  • Standardize FNDC3B detection for potential use as a predictive biomarker for immunotherapy response

  • Develop immunohistochemistry or ELISA protocols suitable for clinical implementation

The strong correlation between FNDC3B expression and immune checkpoint molecules (B7-H3: R = 0.69, PD-L1: R = 0.58) provides a foundation for these research directions.

What novel applications of FNDC3B antibodies are emerging in cancer research?

Emerging applications of FNDC3B antibodies in cancer research include:

• Liquid biopsy development:

  • Detection of circulating FNDC3B or FNDC3B-expressing extracellular vesicles as potential biomarkers

  • Correlation of circulating FNDC3B levels with tumor burden or treatment response

• Antibody-drug conjugates (ADCs):

  • Exploration of FNDC3B as a potential ADC target in cancers with high expression

  • Development of internalizing antibodies against FNDC3B for drug delivery

• Single-cell analysis:

  • Integration of FNDC3B antibodies in mass cytometry (CyTOF) or imaging mass cytometry

  • Exploration of FNDC3B expression heterogeneity at the single-cell level

• 3D culture and organoid applications:

  • Investigation of FNDC3B expression and function in 3D tumor models

  • Use of antibodies to track expression changes during organoid development

• Mechanistic studies of FNDC3B in cancer progression:

  • Identification of FNDC3B interaction partners through co-immunoprecipitation studies

  • Investigation of FNDC3B's role in cancer stem cell maintenance

  • Exploration of FNDC3B's impact on tumor metabolism

These emerging applications expand the utility of FNDC3B antibodies beyond traditional expression studies to mechanistic and translational research.

What methodological advances could improve FNDC3B detection and functional analysis?

Several technological and methodological advances could enhance FNDC3B research:

• Advanced imaging techniques:

  • Super-resolution microscopy for detailed subcellular localization of FNDC3B

  • Live-cell imaging with fluorescently tagged antibody fragments to track FNDC3B dynamics

  • Expansion microscopy to resolve FNDC3B distribution at nanoscale resolution

• Mass spectrometry-based approaches:

  • Development of targeted proteomics assays for absolute quantification of FNDC3B

  • Phosphoproteomics to identify FNDC3B post-translational modifications

  • Proximity labeling combined with mass spectrometry to identify the FNDC3B interactome

• Genetic engineering tools:

  • CRISPR-based endogenous tagging of FNDC3B for live visualization

  • Domain-specific mutations to dissect functional regions of FNDC3B

  • Inducible expression systems to study temporal aspects of FNDC3B function

• Antibody engineering:

  • Development of recombinant antibodies with improved specificity and sensitivity

  • Creation of antibodies specific to post-translationally modified forms of FNDC3B

  • Generation of nanobodies against FNDC3B for improved tissue penetration

• Computational approaches:

  • Machine learning for automated quantification of FNDC3B expression in tissues

  • Integrative multi-omics analysis to contextualize FNDC3B function

  • Structural modeling to predict antibody binding sites and functional domains

These methodological advances would address current limitations in FNDC3B research and enable more sophisticated analyses of its biological functions.