FCN3 Antibody

What is FCN3 protein and what are its key structural features?

FCN3 (also known as H-ficolin or Hakata antigen) is a member of the Ficolin lectin protein family that functions in innate immunity through activation of the lectin complement pathway. The mature human Ficolin-3 protein has the following key features:

• Molecular weight: Approximately 32.9-35 kDa

• Length: 299 amino acid residues in the canonical protein form

• Key domains: N-terminal collagen domain and C-terminal fibrinogen-like domain

• Glycosylation: Contains post-translational modifications including glycosylation

• Oligomerization: Forms trimers through the collagen domain, with larger ~650 kDa, 18-subunit oligomers formed by disulfide links at the N-terminus

FCN3 is primarily expressed in the liver and lungs, with the liver thought to be responsible for circulating FCN3 levels. It binds to specific carbohydrates (including mannose, galactose, and D-fucose) through its fibrinogen-like domain, enabling recognition of microbial patterns .

What applications are FCN3 antibodies commonly used for?

FCN3 antibodies are employed in various research applications, with the most common being:

• Western blotting (WB): For detecting FCN3 protein in cell or tissue lysates

• Enzyme-linked immunosorbent assay (ELISA): For quantifying FCN3 in serum, plasma, or other biological fluids

• Immunohistochemistry (IHC): For analyzing FCN3 expression patterns in tissue sections

• Flow cytometry (FCM): For detecting FCN3 in cell populations

• Immunoprecipitation (IP): For isolating FCN3 protein complexes

Many commercially available antibodies have been validated for multiple applications, but researchers should verify specificity for their particular experimental system.

What specimen types can be analyzed using FCN3 antibodies?

FCN3 antibodies have been validated for use with multiple specimen types:

When analyzing FCN3 in clinical samples, researchers should consider that circulating levels in healthy individuals average around 18 μg/mL, with significant variability possible in disease states .

How can researchers distinguish between different FCN3 isoforms?

Human FCN3 has been reported to have up to 2 different isoforms:

• Isoform 1: Contains an additional 11 amino acids between the collagen and fibrinogen-like domains

• Isoform 2 (canonical): 299 amino acid residues

To distinguish between these isoforms:

• Use isoform-specific antibodies: Select antibodies raised against peptides unique to each isoform.

• Employ high-resolution SDS-PAGE: The slight molecular weight difference can be resolved with 10-12% gels using extended run times.

• RT-PCR with isoform-specific primers: For mRNA expression analysis.

• Mass spectrometry: For definitive identification of protein isoforms in complex samples.

Researchers should be aware that the observed molecular weight may differ from theoretical predictions due to post-translational modifications. FCN3 antibodies have reported detecting bands at both 35 kDa and 50 kDa in Western blotting applications .

What are the methodological considerations when using FCN3 antibodies in cancer research?

FCN3 has been identified as a potential tumor suppressor in several cancers, including lung adenocarcinoma and hepatocellular carcinoma. When investigating FCN3 in cancer contexts:

• Expression analysis considerations:

  • Compare FCN3 levels in paired tumor and non-tumor tissues

  • Assess both mRNA (qRT-PCR) and protein levels (Western blot, IHC)

  • Multiple studies show FCN3 is often downregulated in cancer tissues compared to adjacent normal tissues

• Immunohistochemistry scoring methods:

  • Studies have used systems based on percentages of positive cells:

    • Grade 1: <25% positive cells

    • Grade 2: 25-50% positive cells

    • Grade 3: 51-75% positive cells

    • Grade 4: >75% positive cells

  • Assessment by two independent researchers is recommended to minimize bias

• Functional studies:

  • When overexpressing FCN3 in cancer cell lines, distinguish between effects of secreted and non-secreted forms

  • Studies show ectopic expression of FCN3 can lead to cell cycle arrest and apoptosis via endoplasmic reticulum stress in some cancer types

  • In HCC, FCN3 can promote ferroptosis through downregulation of MUFA synthesis

• Prognostic significance:

  • Correlate FCN3 expression with clinical parameters and survival data

  • Low FCN3 expression has been associated with poor prognosis in several cancers

What are the best practices for validating FCN3 antibody specificity?

Ensuring antibody specificity is crucial for reliable research outcomes. For FCN3 antibodies, recommended validation practices include:

• Positive and negative controls:

  • Positive: Liver tissue/hepatocytes (high FCN3 expression)

  • Negative: Cell lines with confirmed low/no FCN3 expression

  • siRNA knockdown: Compare FCN3 detection in cells with/without FCN3 siRNA treatment

• Cross-reactivity assessment:

  • Test antibody against other Ficolin family members (FCN1, FCN2)

  • Commercial antibodies typically show <10% cross-reactivity with related proteins

• Recombinant protein controls:

  • Use purified recombinant FCN3 as a positive control

  • Confirm molecular weight matches expectations (accounting for tags)

• Multiple detection methods:

  • Confirm FCN3 expression using orthogonal techniques (e.g., Western blot, IHC, and qRT-PCR)

  • Compare results from antibodies targeting different epitopes

• Knockout/overexpression validation:

  • Use FCN3 knockout cell lines or tissues (if available)

  • Test in overexpression systems with tagged FCN3 constructs

How do post-translational modifications affect FCN3 antibody detection?

FCN3 undergoes several post-translational modifications that can impact antibody binding:

• Glycosylation:

  • FCN3 contains multiple glycosylation sites that can alter apparent molecular weight

  • Glycosylation patterns may vary between tissue sources

  • Deglycosylation experiments (using PNGase F or similar enzymes) can help confirm antibody specificity

• Oligomerization:

  • FCN3 forms oligomers via disulfide bonds

  • Non-reducing vs. reducing conditions in Western blotting will show different banding patterns

  • Higher molecular weight bands (~650 kDa) may be detected under non-reducing conditions

• Recommended approach for complex samples:

  • Use reducing agents (β-mercaptoethanol or DTT) to break disulfide bonds

  • Include denaturation steps (boiling in SDS) to disrupt protein complexes

  • Consider deglycosylation treatment for more precise molecular weight determination

What factors affect FCN3 detection in functional complement activation assays?

When studying FCN3's role in complement activation:

• Sample handling considerations:

  • FCN3-mediated complement activation is calcium-dependent

  • Use calcium-containing buffers for functional assays

  • Avoid EDTA which chelates calcium and inhibits complement activation

• ELISA-based functional assays:

  • Acetylated bovine serum albumin (acBSA) can be used as a solid-phase ligand for FCN3

  • Minimal binding of FCN2 and no binding of FCN1 to acBSA allows selective assessment of FCN3

  • Downstream complement activation can be measured through C4, C3, and terminal complement complex (TCC) deposition

• Interference considerations:

  • Classical pathway activation may interfere with FCN3-specific complement activation

  • Chemical inhibitors can be used to block classical pathway interference

  • Dilution series helps identify optimal serum concentration for FCN3-specific activity

• Controls for functional assays:

  • FCN3-depleted serum as negative control

  • Purified FCN3 protein for reconstitution experiments

  • C1q-depleted serum to eliminate classical pathway activation

How can FCN3 antibodies be applied in studying its role in tumor suppression?

Recent research has revealed FCN3's tumor suppressive functions through various mechanisms:

• Cell cycle and apoptosis analysis:

  • FCN3 overexpression in cancer cell lines induces cell cycle arrest and apoptosis

  • Recommended assays: BrdU labeling for proliferation, colony formation assays, flow cytometry for cell cycle analysis

• Signaling pathway investigation:

  • In HCC, FCN3 activates the p53 signaling pathway

  • FCN3 can induce apoptosis via endoplasmic reticulum (ER) stress in lung cancer

  • In experimental designs, include inhibitors of specific pathways to confirm mechanisms

• Cellular localization studies:

  • The fibrinogen domain of FCN3, when localized to ER, is sufficient for inducing apoptosis

  • Use immunofluorescence with organelle markers to track FCN3 localization

  • For protein interaction studies, techniques such as co-immunoprecipitation, FRET, and proximity ligation assay can be employed

• In vivo tumor models:

  • Xenograft models with FCN3-overexpressing cancer cells show reduced tumor growth

  • Sample collection timeline: 4-5 weeks for significant differences in tumor volume

  • EdU injection (50 mg/kg) 4 hours prior to tissue collection for proliferation assessment

• Molecular mechanism investigation:

  • Protein-protein interaction: FCN3 binds to SBDS, affecting EIF6 nuclear translocation

  • Studies should include both overexpression and knockdown approaches

  • Nucleocytoplasmic separation assays can reveal changes in protein localization

What are the key considerations for using FCN3 antibodies in hepatocellular carcinoma research?

Recent research has identified important roles of FCN3 in hepatocellular carcinoma (HCC):

What is the optimal procedure for using FCN3 antibodies in immunohistochemistry?

For successful FCN3 immunohistochemistry on tissue sections:

• Tissue preparation:

  • Formalin-fixed, paraffin-embedded (FFPE) sections (4-5 μm thickness)

  • Deparaffinization in xylene followed by rehydration through graded alcohols

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) is typically effective

  • Alternative: EDTA buffer (pH 9.0) if citrate buffer yields weak signals

• Blocking and antibody incubation:

  • Block endogenous peroxidase activity (3% H₂O₂, 10 minutes)

  • Block non-specific binding (5% BSA or normal serum, 1 hour)

  • Primary antibody dilution: typically 1:100 to 1:500 (optimize for specific antibody)

  • Incubation: overnight at 4°C or 1-2 hours at room temperature

• Detection system:

  • HRP-conjugated secondary antibody or streptavidin-biotin system

  • Visualization with DAB (3,3'-diaminobenzidine) chromogen (5 minutes)

  • Counterstain with hematoxylin (5 minutes)

  • Dehydration and mounting with permanent medium

• Evaluation of FCN3 staining:

  • Assess both intensity and percentage of positive cells

  • FCN3 is typically detected in cytoplasm or both cytoplasm and partial nuclear areas

  • Non-tumor liver tissues consistently show strong immunostaining (3+ to 4+)

What are the recommended protocols for FCN3 ELISA assays?

For quantitative measurement of FCN3 in human samples:

• Sample collection and preparation:

  • Serum or plasma (heparin preferred over EDTA for functional studies)

  • Typical dilution ranges: 1:100 to 1:1000 (optimize based on expected concentration)

  • Average FCN3 concentration in healthy human serum: ~18 μg/mL

• ELISA procedure:

  • Pre-coated plates with anti-FCN3 capture antibody

  • Sample addition and incubation (typically 100 μL per well)

  • Detection with biotinylated anti-FCN3 antibody

  • Signal development with streptavidin-HRP and chromogenic substrate

  • Absorbance reading at 450 nm

• Standard curve:

  • Commercial kits typically provide detection ranges of:

    • 78-5000 pg/mL

    • 0.156-10 ng/mL

    • 7.8-500 ng/mL (varies by manufacturer)

• Quality control:

  • Include positive and negative controls

  • Run samples in duplicate

  • Consider including known concentration samples to verify assay performance

• Functional complement activation assays:

  • Use acetylated BSA (acBSA) as a solid-phase ligand for FCN3

  • Measure downstream complement components (C4, C3, TCC) to assess functional activity

  • Ensure calcium-dependent binding by including appropriate controls

How should researchers troubleshoot inconsistent FCN3 antibody results?

When encountering inconsistent results with FCN3 antibodies:

• Storage and handling issues:

  • Ensure proper storage conditions (-20°C for most antibodies)

  • Avoid repeated freeze-thaw cycles (aliquot antibodies upon receipt)

  • Check antibody expiration dates and visible precipitation

• Sample-related factors:

  • Protein degradation: Use fresh samples and add protease inhibitors

  • Improper sample preparation: Ensure complete lysis for cell/tissue samples

  • Interfering substances: Consider sample clean-up procedures

• Protocol optimization:

  • Titrate antibody concentration (typical dilutions for Western blot: 1:500-1:2000)

  • Adjust incubation time and temperature

  • Modify blocking conditions (try different blocking agents)

  • For Western blots, test different membrane types (PVDF vs. nitrocellulose)

• Detection system issues:

  • Test alternative secondary antibodies

  • For chemiluminescent detection, try different exposure times

  • Consider more sensitive detection systems for low-abundance targets

• Epitope accessibility problems:

  • Try different antigen retrieval methods for IHC

  • Test antibodies targeting different epitopes

  • For Western blotting, try both reducing and non-reducing conditions

What considerations are important when designing FCN3 knockdown or overexpression experiments?

For genetic manipulation of FCN3 expression:

• Knockdown approaches:

  • siRNA: Studies have successfully used siRNA targeting FCN3 (siFCN3)

  • Multiple siRNA sequences should be tested to confirm specificity

  • Typical knockdown verification: 24-72 hours post-transfection via qRT-PCR and Western blot

• Overexpression strategies:

  • Full-length FCN3 with C-terminal tag (His, Myc) for detection

  • Consider domain-specific constructs (fibrinogen domain shown to be sufficient for some functions)

  • Vector selection: pcDNA3.1(+) has been successfully used

• Transfection considerations:

  • Cell line-specific optimization of transfection reagents

  • Lipofectamine 3000 for plasmid transfection

  • Lipofectamine 2000 for siRNA transfection

  • Verify expression/knockdown before functional assays

• Functional readouts:

  • For cancer studies: proliferation, apoptosis, cell cycle analysis

  • For complement studies: C4/C3 deposition, complement activation

  • For protein interaction studies: co-immunoprecipitation, FRET

• Special considerations:

  • Distinguish between effects of secreted vs. intracellular FCN3

  • For FRET assays, FCN3 and interacting partners can be cloned into pAcGFP1 and pDsRedMonomer vectors

  • For xenograft models, typically 5×10⁶ cells in 150μL DMEM are injected subcutaneously

How is FCN3 research advancing our understanding of cancer biology?

Recent discoveries about FCN3's role in cancer include:

• Novel tumor suppressor functions:

  • FCN3 acts as a tumor suppressor in lung adenocarcinoma through ER stress induction

  • The fibrinogen domain, when not secreted and localized to ER, induces apoptosis

  • Downregulation of FCN3 correlates with increased mortality in lung adenocarcinoma patients

• Hepatocellular carcinoma mechanisms:

  • FCN3 promotes ferroptosis in HCC cells

  • Mechanistically, FCN3 directly binds to insulin receptor β (IR-β)

  • This inhibits IR-β phosphorylation and suppresses SREBP1c expression

  • Results in downregulation of de novo lipogenesis and reduced MUFA levels

• Prognostic significance:

  • Low FCN3 expression associates with poor prognosis in multiple cancers

  • In HCC, low FCN3 correlates with clinicopathological features including:

    • AFP levels

    • Tumor size and number

    • Microvascular invasion

    • Edmondson-Steiner classification

• p53 pathway interactions:

  • FCN3 modulates nuclear translocation of eukaryotic initiation factor 6 (EIF6)

  • This occurs through binding to ribosome maturation factor (SBDS)

  • Induces ribosomal stress and activation of the p53 pathway

  • Forms a p53/YBX1/SBDS negative feedback loop

What is the emerging role of FCN3 in disease diagnosis and prognosis?

Research indicates FCN3 has potential as a biomarker in various conditions:

How do technological advances impact FCN3 antibody applications in research?

Emerging technologies enhancing FCN3 research include:

• Advanced imaging techniques:

  • Super-resolution microscopy enables detailed subcellular localization of FCN3

  • Confocal laser scanning microscopy for FRET assays evaluating FCN3 protein interactions

  • Multiplexed immunofluorescence for examining FCN3 in relation to other markers

• Protein interaction studies:

  • FRET using tagged constructs (AcGFP1-FCN3 and DsRedMonomer-SBDS)

  • Co-immunoprecipitation with tagged proteins (His-FCN3, Myc-SBDS)

  • Nucleocytoplasmic separation assay for studying compartmentalization

• Metabolomic integration:

  • Combined analysis of FCN3 expression with lipid metabolism

  • Metabonomic analysis to assess intracellular and tissue lipid levels

  • Links between FCN3, ferroptosis, and MUFA synthesis

• Single-cell applications:

  • Single-cell RNA sequencing to examine FCN3 expression heterogeneity

  • Mass cytometry for protein-level analysis in complex cell populations

  • Spatial transcriptomics to map FCN3 expression in tissue microenvironments

• CRISPR-based approaches:

  • CRISPR/Cas9 for creating FCN3 knockout cell lines and animal models

  • CRISPRi for targeted FCN3 repression

  • CRISPR activation systems for enhancing endogenous FCN3 expression

These technological advances provide researchers with more precise tools for studying FCN3's roles in normal physiology and disease pathology.