FAM20A Antibody

What is FAM20A and what are its key biological functions?

FAM20A is a golgi-associated secretory pathway pseudokinase with a canonical protein length of 541 amino acid residues and a mass of 61.4 kDa in humans. Despite lacking kinase activity, it functions as an allosteric activator of the Golgi serine/threonine protein kinase FAM20C. FAM20A is primarily localized in the endoplasmic reticulum and Golgi apparatus, though it can also be secreted . It plays a critical role in biomineralization processes, particularly in tooth development. The protein undergoes post-translational modifications, notably N-glycosylation, which may regulate its function and interactions. In research contexts, FAM20A is often studied in relation to amelogenesis imperfecta, gingival fibromatosis, and ectopic calcification disorders, where mutations in the gene have been implicated .

Which tissue types typically express FAM20A at significant levels?

FAM20A demonstrates varying expression patterns across tissues, with notably high expression reported in the lung and liver . At the cellular level, single-cell sequencing analysis has revealed that FAM20A is specifically expressed in alveolar cells within lung tissue. This expression pattern is altered in pathological conditions - particularly in lung squamous cell carcinoma (LUSC), where FAM20A transcription levels are significantly reduced in tumor cells compared to healthy controls . Understanding these tissue-specific expression patterns is essential when designing experiments involving FAM20A antibodies, particularly when selecting appropriate positive and negative control tissues for validation.

How does FAM20A relate to other members of the FAM20 protein family?

FAM20A belongs to the Family with Sequence Similarity 20 (FAM20) protein family, with its closest functional relationship being to FAM20C. Structurally, they share significant homology, but FAM20A functions as a pseudokinase lacking catalytic activity. Instead, it serves as an allosteric activator of FAM20C, enhancing its kinase activity toward secreted phosphoproteins. This relationship has been conclusively demonstrated through studies of knockout mice and patients with mutations in either gene . Both proteins are involved in biomineralization processes, and mutations in either can lead to similar clinical manifestations including amelogenesis imperfecta, gingival fibromatosis, and ectopic calcification. When designing experiments targeting FAM20A, researchers should be mindful of this close relationship and implement controls that can distinguish between these family members.

What are the optimal conditions for FAM20A immunodetection in different experimental applications?

For Western blot applications, FAM20A antibodies have demonstrated reliable detection across a wide range of species, including human, mouse, rabbit, rat, bovine, dog, guinea pig, horse, and pig samples . When performing Western blot analysis:

• Use 20-30 μg of total protein lysate from tissues with known FAM20A expression (lung or liver) as positive controls

• Include recombinant FAM20A protein standards where possible

• Be aware that the detected molecular weight may vary from the predicted 61.4 kDa due to post-translational modifications, particularly N-glycosylation

For immunohistochemistry applications:

• Antigen retrieval is typically required (citrate buffer pH 6.0 provides consistent results)

• Blocking with 5-10% normal serum from the same species as the secondary antibody for 1-2 hours reduces background

• Primary antibody concentrations between 1:100 and 1:500 dilutions typically yield optimal staining, though this should be empirically determined for each antibody

• Include appropriate tissue controls, particularly lung tissue sections where FAM20A expression has been well-characterized

How can researchers validate FAM20A antibody specificity for experimental applications?

A multi-faceted approach to validating FAM20A antibody specificity is recommended:

• Genetic controls: Include FAM20A knockout or knockdown samples alongside wildtype samples to confirm signal disappearance

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to demonstrate signal blocking

• Multiple antibody validation: Employ antibodies targeting different epitopes of FAM20A to cross-validate findings

• Orthogonal methods: Confirm protein detection with mRNA expression data using qPCR or RNA-seq

• Cross-reactivity assessment: Test against closely related family members, particularly FAM20C, to ensure specificity

These validation steps are particularly crucial when studying FAM20A in lung cancer contexts, where expression changes may be subtle but biologically significant .

What protocols maximize detection sensitivity when FAM20A expression is downregulated in cancer samples?

The significant downregulation of FAM20A in lung squamous cell carcinoma presents a methodological challenge for researchers . To maximize detection sensitivity:

• Signal amplification systems: Consider using tyramide signal amplification or polymer-based detection systems that can enhance sensitivity by 10-100 fold

• Extended primary antibody incubation: Overnight incubation at 4°C can improve antigen detection in low-expression samples

• Optimized sample preparation: For protein extraction, use buffers containing phosphatase inhibitors to preserve post-translational modifications that may affect antibody recognition

• Batch processing: Process paired normal and tumor samples simultaneously to minimize technical variation

• Quantitative image analysis: Employ digital pathology tools with appropriate controls to detect subtle expression differences

These approaches are particularly valuable when using FAM20A as a potential diagnostic biomarker in LUSC, where precise quantification may have clinical significance.

How does FAM20A expression correlate with immune infiltration in tumor microenvironments?

FAM20A expression demonstrates significant correlations with immune cell populations in the tumor microenvironment, particularly in lung squamous cell carcinoma:

• FAM20A expression inversely correlates with tumor purity in LUSC

• FAM20A shows positive correlations with multiple immune cell types, including:

  • B cells

  • CD8+ and CD4+ T cells

  • Macrophages

  • Neutrophils

  • Dendritic cells (conventional, plasmacytoid, and activated)

This suggests that reduced FAM20A expression in LUSC might contribute to tumor immune evasion mechanisms. Researchers using FAM20A antibodies in cancer immunology studies should consider analyzing these immune populations concurrently.

What evidence supports FAM20A as a diagnostic biomarker for lung squamous cell carcinoma?

Multiple lines of evidence support FAM20A's potential as a diagnostic biomarker for LUSC:

• Expression analysis: FAM20A expression is significantly reduced in LUSC compared to normal lung tissue across multiple datasets (TCGA, GTEx, and CGGA databases)

• Specificity: FAM20A downregulation appears to be selective for LUSC among lung cancer subtypes

• Cellular specificity: Single-cell sequencing demonstrates that FAM20A is expressed in normal alveolar cells but drastically reduced in tumor cells

• Correlation with pathways: FAM20A expression negatively correlates with DNA repair signaling pathways, potentially contributing to radiotherapy resistance

• Epigenetic regulation: FAM20A hypermethylation is associated with poor LUSC prognosis

What methodological approaches can detect FAM20A's interaction with DNA repair pathways?

To investigate FAM20A's relationship with DNA repair pathways in cancer research:

• Co-immunoprecipitation: Use FAM20A antibodies to pull down protein complexes and probe for DNA repair components

• Proximity ligation assays: Detect in situ protein-protein interactions between FAM20A and DNA repair proteins

• ChIP-seq analysis: Determine if FAM20A associates with chromatin at sites of DNA damage or repair

• γH2AX foci quantification: Measure DNA damage repair efficiency in cells with modulated FAM20A expression

• Radiotherapy response assays: Compare colony formation after irradiation in cells with varying FAM20A expression

The negative correlation between FAM20A and key DNA repair proteins suggests a potential mechanism for radiotherapy resistance in low-FAM20A LUSC tumors . This presents an important area for further investigation with significant therapeutic implications.

How can researchers distinguish between intracellular and secreted FAM20A forms?

FAM20A exhibits dual localization - intracellular (ER and Golgi) and extracellular (secreted) . To distinguish between these pools:

• Cellular fractionation: Separate membrane-bound organelles from cytosolic and secreted fractions, followed by Western blot analysis

• Immunofluorescence with compartment markers: Co-stain with organelle markers (e.g., GM130 for Golgi) to determine intracellular localization

• Cell surface biotinylation: Label and isolate cell surface proteins to detect membrane-associated FAM20A

• Conditioned media analysis: Collect and concentrate cell culture supernatants to detect secreted FAM20A

• Glycosylation analysis: Treat samples with glycosidases to identify differentially glycosylated forms associated with secretory pathways

Researchers should be aware that post-translational modifications, particularly N-glycosylation, may affect antibody recognition differentially between intracellular and secreted forms, potentially requiring different antibodies or detection methods for each pool.

What experimental designs best demonstrate FAM20A-FAM20C functional interactions?

To investigate the functional relationship between FAM20A and FAM20C:

• Sequential immunoprecipitation: Use antibodies against FAM20A to pull down complexes, followed by Western blot for FAM20C

• In vitro kinase assays: Measure FAM20C kinase activity with and without recombinant FAM20A

• Co-expression studies: Modulate FAM20A expression and measure changes in FAM20C activity or localization

• Structural studies: Employ antibodies for protein purification for crystallography or cryo-EM studies

• Proximity-dependent labeling: Use BioID or APEX2 fusions to identify proteins in the immediate vicinity of FAM20A

Given the established relationship where FAM20A functions as an allosteric activator of FAM20C , these approaches can help elucidate the molecular details of this interaction and identify potential sites for therapeutic intervention.

How can FAM20A antibodies contribute to understanding its role in tumor immune evasion?

Based on the positive correlation between FAM20A expression and immune cell infiltration , researchers can employ FAM20A antibodies to:

• Multiplex immunohistochemistry: Co-stain tumor sections for FAM20A and immune cell markers to analyze spatial relationships

• Flow cytometry: Quantify FAM20A in tumor cells alongside immune population analysis

• Single-cell protein analysis: Employ CyTOF or single-cell Western approaches to correlate FAM20A with immune checkpoints at the single-cell level

• In vitro co-culture assays: Manipulate FAM20A expression in tumor cells and measure changes in immune cell activation, migration, or cytotoxicity

• Extracellular vesicle isolation: Determine if secreted FAM20A in exosomes affects immune cell function

Understanding how reduced FAM20A in LUSC might contribute to immune evasion could reveal new immunotherapeutic strategies. The observed positive correlation between FAM20A and immune checkpoint molecules like CTLA-4 suggests potential relevance to checkpoint inhibitor therapy response .

What techniques can identify how FAM20A epigenetic regulation impacts gene expression in cancer?

To investigate the epigenetic regulation of FAM20A in cancer:

• Bisulfite sequencing: Quantify methylation at specific CpG sites in the FAM20A promoter

• Chromatin immunoprecipitation (ChIP): Identify histone modifications and transcription factor binding at the FAM20A locus

• Methylation-specific PCR: Develop targeted assays for clinical implementation

• CRISPR epigenetic editing: Modulate specific epigenetic marks at the FAM20A locus to establish causality

• DNA methyltransferase inhibitor studies: Treat cells with agents like 5-azacytidine to determine if FAM20A expression can be restored

The observation that FAM20A hypermethylation correlates with poor prognosis in LUSC suggests that epigenetic silencing is a key regulatory mechanism. Developing methodologies to accurately detect these epigenetic changes could complement protein-based approaches using FAM20A antibodies.