FAM20C Antibody

What is FAM20C and what are its key functions in cellular biology?

FAM20C (Family with sequence similarity 20 member C) is a Golgi-associated secretory pathway kinase that phosphorylates secreted proteins by recognizing the protein motif "Ser-x-Glu/phospho-Ser." The 584 amino acid protein (66.2 kDa) functions as a calcium-binding kinase that phosphorylates caseins and several secreted proteins involved in biomineralization, including secretory calcium binding phosphoproteins (SCPPs). FAM20C plays critical roles in biomineralization, lipid homeostasis, cell adhesion, and migration. Many FAM20C substrates are directly related to tumor cell apoptosis and metastasis, including insulin-like growth factor binding proteins, osteopontin, and serine protease inhibitors . The protein is widely expressed across various tissues and has orthologs in multiple species including canine, porcine, monkey, mouse and rat .

What are the common alternative names for FAM20C in scientific literature?

When researching FAM20C, it's important to be aware of its alternative nomenclature to ensure comprehensive literature searches. FAM20C is also known as:

• Family with sequence similarity 20 member C

• DMP4 (Dentin matrix protein 4)

• DMP-4

• G-CK

• GEF-CK

• Extracellular serine/threonine protein kinase FAM20C

• Golgi-enriched fraction casein kinase

What criteria should researchers use when selecting an appropriate FAM20C antibody for specific applications?

When selecting a FAM20C antibody, researchers should consider several key parameters based on experimental requirements:

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IHC, ICC, IF, ELISA). For example, antibody catalog number 25395-1-AP has been validated for IHC applications with a recommended dilution of 1:50-1:500 .

• Species reactivity: Confirm cross-reactivity with your species of interest. Many commercial FAM20C antibodies show reactivity with human, mouse, and rat samples .

• Epitope recognition: Consider whether the antibody targets specific regions (e.g., C-terminal, middle region) which may affect recognition of splice variants or processed forms.

• Validation data: Review published validation data such as immunohistochemical staining patterns in positive control tissues like human kidney or ovary .

• Clonality: Polyclonal antibodies often provide higher sensitivity while monoclonal antibodies offer greater specificity.

• Conjugation: Determine if your application requires unconjugated antibodies or conjugates (e.g., biotin, APC).

How can researchers validate the specificity of FAM20C antibodies for their experimental systems?

Validation of FAM20C antibody specificity requires multiple complementary approaches:

• Positive controls: Use tissues known to express FAM20C (e.g., human kidney and ovary tissues have been validated for IHC with catalog 25395-1-AP) .

• Blocking peptide competition: Pre-incubate the antibody with the immunizing peptide to confirm signal reduction in target applications.

• Knockdown/knockout validation: Compare staining between wildtype and FAM20C-deficient samples (gene silencing via siRNA or CRISPR).

• Multiple antibody comparison: Test different antibodies targeting distinct FAM20C epitopes to confirm consistent localization patterns.

• Western blot molecular weight verification: Confirm detection at the expected molecular weight (66.2 kDa for full-length FAM20C) .

• Subcellular localization concordance: Verify that the observed subcellular localization matches expected patterns (Golgi apparatus and secretory pathway).

What are the optimal protocols for immunohistochemical detection of FAM20C in formalin-fixed paraffin-embedded tissues?

For optimal IHC detection of FAM20C in FFPE tissues, the following protocol is recommended based on validated antibody performance:

• Section preparation: Cut 4 μm-thick paraffin sections and mount on positively charged slides.

• Antigen retrieval: Perform heat-induced epitope retrieval (HIER) with TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0 as an alternative .

• Blocking: Block endogenous peroxidase activity with 3% H₂O₂, followed by protein blocking with 5-10% normal serum.

• Primary antibody incubation: Apply FAM20C antibody at 1:50-1:500 dilution (antibody-dependent) and incubate at 4°C overnight or at room temperature for 1-2 hours .

• Detection system: Use a high-sensitivity detection system such as polymer-based detection (e.g., non-biotin universal two-step immunohistochemistry kit) .

• Visualization: Develop with DAB and counterstain with hematoxylin.

• Interpretation: FAM20C positive expression appears as brown-yellow particles in the nucleus and cytoplasm. Quantification can be performed using the histochemical score (H-score) method as described in published protocols .

How should FAM20C subcellular localization patterns be interpreted in immunofluorescence studies?

When interpreting FAM20C localization in immunofluorescence studies:

What is the significance of FAM20C overexpression in cancer research, particularly in lower-grade gliomas?

FAM20C overexpression has emerged as a significant biomarker in cancer research, with particular relevance to lower-grade gliomas (LGG):

How can FAM20C phosphorylation activity be measured in relation to its substrates in cancer cells?

Assessing FAM20C kinase activity toward its substrates in cancer cells requires specialized methodological approaches:

• Phospho-specific antibody detection: Use antibodies specifically recognizing phosphorylated forms of known FAM20C substrates (e.g., phospho-osteopontin, phospho-IGFBP7).

• In vitro kinase assays: Immunoprecipitate FAM20C from cancer cells and perform in vitro kinase reactions with purified substrates, followed by detection of phosphorylation using:

  • ³²P-ATP incorporation

  • Phospho-specific antibodies

  • Mass spectrometry

• Phosphoproteomics approach: Compare phosphopeptide profiles between:

  • FAM20C-overexpressing cells

  • FAM20C-depleted cells

  • FAM20C inhibitor-treated cells
    focusing on the "Ser-x-Glu/phospho-Ser" motif recognized by FAM20C

• Substrate-specific reporters: Develop FRET-based biosensors incorporating known FAM20C substrate sequences to monitor phosphorylation dynamics in live cells.

• Functional correlation: Correlate phosphorylation status of specific substrates with cancer-relevant phenotypes such as cell migration, invasion, and apoptosis resistance to establish functional significance.

How do post-translational modifications affect FAM20C function and antibody recognition?

Post-translational modifications (PTMs) of FAM20C have significant implications for both its biological function and experimental detection:

• Auto-phosphorylation: As a kinase, FAM20C can undergo auto-phosphorylation, which may regulate its activity. Antibodies raised against unmodified epitopes may show reduced binding to heavily phosphorylated protein.

• Glycosylation: As a secretory pathway protein, FAM20C undergoes glycosylation which can affect:

  • Protein stability and trafficking

  • Molecular weight detection in Western blots (appearing larger than the calculated 66.2 kDa)

  • Epitope accessibility for certain antibodies

• Proteolytic processing: Evidence suggests FAM20C may undergo processing during secretion or activation. Antibodies targeting different regions (N-terminal, middle region, C-terminal) may yield different detection patterns based on processing status.

• Calcium binding: FAM20C function is calcium-dependent, and conformational changes upon calcium binding may expose or mask epitopes recognized by specific antibodies.

• Experimental considerations: Researchers should consider using phosphatase treatments, deglycosylation enzymes, or denaturing conditions when facing inconsistent antibody recognition to determine if PTMs are affecting detection.

What approaches are recommended for studying FAM20C-substrate interactions in the context of the tumor microenvironment?

Investigating FAM20C-substrate interactions within the complex tumor microenvironment requires specialized methodological strategies:

• Proximity ligation assays (PLA): Detect direct interactions between FAM20C and potential substrates in tissue sections while preserving spatial context.

• Secretome analysis: Compare phosphoproteome profiles of secreted proteins from tumor and stromal cells with and without FAM20C modulation (overexpression, knockdown, inhibition).

• 3D co-culture systems: Establish 3D co-culture models incorporating cancer cells, fibroblasts, immune cells, and endothelial cells to study FAM20C-substrate dynamics in a more physiologically relevant context.

• Ex vivo tissue explant cultures: Maintain tumor tissue architecture while manipulating FAM20C expression or activity to study substrate phosphorylation in near-native conditions.

• In situ phosphorylation mapping: Combine laser capture microdissection with mass spectrometry to map phosphorylation patterns of FAM20C substrates in different tumor regions (core, invasive front, surrounding stroma).

• Multiplexed immunofluorescence: Simultaneously visualize FAM20C, potential substrates, phosphorylated substrates, and cell-type markers to understand spatial relationships in the tumor microenvironment.

What are the common challenges in Western blot detection of FAM20C and how can they be addressed?

Western blot detection of FAM20C can present several technical challenges that require specific optimization strategies:

How can researchers optimize antibody-based detection of FAM20C in cells with variable expression levels?

Optimizing FAM20C detection across samples with varying expression levels requires careful methodological considerations:

• Titration experiments: Perform antibody dilution series across samples with known variable expression to identify optimal concentration that detects low expression without saturating high expression samples.

• Signal amplification strategies:

  • For IHC/ICC: Use polymer-based detection systems or tyramide signal amplification for enhanced sensitivity

  • For flow cytometry: Consider secondary antibodies with higher fluorophore conjugation ratios

  • For Western blot: Employ enhanced chemiluminescence substrates designed for high sensitivity

• Exposure/acquisition optimization:

  • For Western blot: Capture multiple exposure times to ensure linearity of signal

  • For fluorescence: Adjust acquisition parameters (exposure time, gain) based on control samples

  • For IHC: Standardize development times based on positive controls

• Quantification approaches:

  • Use appropriate internal loading controls

  • Consider normalizing to total protein methods (Ponceau, REVERT, etc.)

  • Employ digital image analysis with appropriate dynamic range

• Validation strategies:

  • Include samples with known FAM20C expression levels (overexpression, knockdown)

  • Consider orthogonal detection methods (mRNA level verification by qPCR)

How might emerging technologies advance our understanding of FAM20C function beyond current antibody-based approaches?

Emerging technologies offer promising approaches to expand our understanding of FAM20C biology beyond traditional antibody methods:

• CRISPR-based tagging: Endogenous tagging of FAM20C with fluorescent proteins or affinity tags enables:

  • Live-cell imaging of FAM20C dynamics

  • Proximity-dependent labeling to identify interaction partners

  • Chromatin immunoprecipitation studies if nuclear functions are suspected

• Proximity labeling technologies: BioID or APEX2 fused to FAM20C can identify proximal proteins in living cells, revealing potential substrates and interaction partners with temporal and spatial resolution.

• Single-cell phosphoproteomics: Analyze FAM20C-dependent phosphorylation events at single-cell resolution to understand heterogeneity in FAM20C function across different cell populations within complex tissues.

• Nanobody development: Engineer FAM20C-specific nanobodies that can:

  • Track FAM20C in living cells

  • Inhibit specific functions

  • Be used for super-resolution microscopy

• Structural biology approaches: Cryo-EM and X-ray crystallography studies of FAM20C in complex with substrates can provide detailed mechanistic insights into substrate recognition and catalytic function.

What are the key considerations for developing and validating FAM20C inhibitors for potential therapeutic applications?

Development of FAM20C inhibitors for therapeutic applications requires systematic approaches with several key considerations:

• Target validation:

  • Confirm FAM20C dependency in disease models through genetic approaches (knockdown/knockout)

  • Establish clear relationship between FAM20C activity and disease phenotypes

  • Identify patient populations most likely to benefit based on FAM20C expression/activity profiles

• Inhibitor screening and optimization:

  • Develop robust in vitro kinase assays using physiologically relevant substrates

  • Screen diverse chemical libraries for FAM20C inhibition

  • Optimize lead compounds for potency, selectivity, and drug-like properties

• Selectivity profiling:

  • Assess activity against related kinases, particularly other FAM20 family members

  • Perform kinome-wide selectivity profiling to identify off-target effects

  • Evaluate effects on phosphorylation of known FAM20C substrates versus other phosphoproteins

• Cellular and in vivo validation:

  • Confirm target engagement in cellular contexts

  • Evaluate phenotypic effects in disease-relevant cell and animal models

  • Assess pharmacokinetics, pharmacodynamics, and toxicology profiles

• Biomarker development:

  • Identify phosphorylation events that reliably indicate FAM20C inhibition

  • Develop assays to monitor target engagement in clinical samples

  • Establish predictive biomarkers for patient stratification