FAM110C Antibody, FITC conjugated

What is FAM110C and what is its significance in cancer research?

FAM110C (Family with Sequence Similarity 110 Member C) is a cell fate-related gene with emerging importance in cancer research. It plays several critical cellular roles:

• Inhibits cell proliferation by inducing G1/S arrest

• Interacts with the microtubule cytoskeleton to regulate cell morphology

• Suppresses cell migration by inhibiting AKT signaling pathway

• Activates ATM and NHEJ signaling pathways through interaction with HMGB1

• Functions as a potential tumor suppressor in pancreatic cancer by regulating DNA damage repair

How does FITC conjugation work and what are its advantages for antibody labeling?

FITC (Fluorescein Isothiocyanate) conjugation involves the covalent attachment of FITC molecules to antibodies through a chemical reaction between the isothiocyanate group of FITC and primary amines (typically lysine residues) on the antibody, forming stable thiourea bonds.

Conjugation process:

• The antibody is incubated with FITC under slightly alkaline conditions (optimal pH 8.3-9.0)

• A modifier reagent is added to optimize reaction conditions

• The reaction proceeds at room temperature for 3-24 hours in the dark

• A quencher may be added to stop the reaction

Advantages of FITC as a fluorophore:

• High quantum efficiency producing bright green fluorescence

• Excitation/emission maxima at 495/519 nm compatible with standard microscopy and flow cytometry equipment

• Well-established protocols and controls

• Stability of conjugates when properly stored

• Cost-effectiveness compared to newer fluorophores1

The F/P (fluorophore to protein) ratio is critical for optimal performance and is experimentally determined by spectrophotometric analysis or flow cytometric assessment .

What are the common applications of FITC-conjugated FAM110C antibodies?

FITC-conjugated FAM110C antibodies are valuable tools in various research applications:

These applications have helped establish FAM110C's differential roles across cancer types and its potential utility as a biomarker.

What are the optimal storage conditions for FITC-conjugated antibodies?

Proper storage is critical for maintaining the fluorescence and binding activity of FITC-conjugated antibodies:

Short-term storage (up to 6 months):

• Store at 4°C in an amber vial or wrapped in aluminum foil

• Protect from exposure to light at all times

• Include preservatives such as 0.01% sodium azide in storage buffer

Long-term storage:

• Aliquot the antibody to avoid repeated freeze-thaw cycles

• Store at -20°C or -80°C

• Protect from light exposure

• Include cryoprotectants if freezing

Important precautions:

• Continuous exposure to light causes gradual loss of fluorescence

• Repeated freezing and thawing should be strictly avoided as it results in loss of antibody activity

• Before using stored antibodies, centrifuge briefly to collect solution at the bottom of the vial

• When diluting from stock, use high-quality, sterile buffer solutions

Most manufacturers guarantee FITC-conjugated antibodies for six months from the date of receipt when properly stored according to these guidelines.

What controls should be included when using FAM110C-FITC antibodies?

Proper controls are essential for interpreting results with FAM110C-FITC antibodies:

Including these controls helps distinguish true FAM110C signal from technical artifacts and enables accurate interpretation of experimental results across different cancer models.

How do FAM110C expression patterns differ across cancer types, and what are the implications for diagnostic applications?

FAM110C exhibits strikingly different expression patterns and biological roles across cancer types, with important implications for its use as a diagnostic marker:

Pancreatic Ductal Adenocarcinoma (PDAC):

• FAM110C is methylated in 72.89% (207/284) of PDAC cases

• Methylation progressively increases from precursor lesions (IPMN: 41.18%, MCN: 46.67%) to PDAC (p=0.0001, p=0.0389)

• Functions as a tumor suppressor by activating ATM and NHEJ signaling

• Methylation correlates with tumor size (p=0.0103) and serves as an independent prognostic marker for poor survival (p=0.0159)

• Loss of FAM110C sensitizes PDAC cells to ATR/CHK1 inhibitors, suggesting therapeutic vulnerability

Glioblastoma (GBM):

• FAM110C is highly expressed in wild-type IDH1 GBM compared to IDH1-mutant GBM (p=0.0053)

• Higher expression correlates with worse prognosis (p=0.03, HR=1.55)

• Expression predicts survival with increasing accuracy over time (ROC-1 year: 0.647; ROC-3 years: 0.709; ROC-5 years: 0.932)

• Promotes glioma cell migration and invasion

• Knockdown inhibits growth of wild-type GBM cells and reduces migration

Diagnostic implications and considerations:

• Cancer-specific interpretation is essential - high expression is detrimental in GBM but protective in PDAC

• Combined epigenetic and protein-level analysis provides more comprehensive diagnostic information

• For PDAC, FAM110C methylation status may identify patients who could benefit from ATR/CHK1 inhibitors

• For GBM, quantitative assessment of FAM110C expression could help predict patient outcomes

• IDH1 mutation status should be considered alongside FAM110C expression in GBM diagnosis

These contrasting roles underscore the importance of cancer-specific protocols when developing FAM110C-based diagnostic tools.

What methodological approaches can optimize FITC-conjugated FAM110C antibody staining for immunofluorescence?

Optimizing FITC-conjugated FAM110C antibody staining requires careful attention to several key methodological parameters:

Sample preparation:

• Fixation method significantly impacts FAM110C detection - methanol fixation works well for cell lines, but test multiple methods for tissue samples

• For FFPE tissue sections, antigen retrieval methods may be necessary to unmask epitopes

• Permeabilization optimization is critical as FAM110C localizes to both nucleus and cytoplasm

Blocking and antibody incubation:

• Block with PBS containing 10% fetal bovine serum for at least 20 minutes at room temperature

• Dilute FITC-conjugated antibody 1:500 in blocking solution (empirically determine optimal dilution)

• Incubate for 1 hour at room temperature in the dark

• Wash thoroughly with PBS (2 × 5 minutes)

Signal optimization strategies:

• Titrate antibody concentration to determine optimal signal-to-noise ratio

• For low-abundance targets, consider signal amplification systems compatible with FITC

• Use confocal microscopy with appropriate filter sets (excitation ~495nm, emission ~519nm)

• Employ Z-stack acquisition to capture complete subcellular distribution

• Use mounting media with anti-fade agents to reduce photobleaching

FAM110C-specific considerations:

• In PDAC studies, correlate staining patterns with methylation status

• In GBM studies, compare wild-type versus IDH1-mutant samples

• Include appropriate subcellular markers to confirm nuclear/cytoplasmic distribution

• When examining cell cycle effects, synchronize cells by serum withdrawal (12h) followed by serum re-addition

Implementing these optimizations will enhance detection sensitivity and specificity when studying FAM110C across different experimental systems.

How can methylation status of FAM110C be integrated with antibody-based detection for comprehensive analysis?

The inverse relationship between FAM110C methylation and protein expression necessitates an integrated approach for comprehensive analysis:

Integrated methodological workflow:

• Methylation analysis:

  • Perform methylation-specific PCR (MSP) using primers targeting the FAM110C promoter region

  • Validate methylation density through bisulfite sequencing (BSSQ)

  • Use normal lymphocyte DNA (NL) as unmethylation control and in vitro methylated DNA (IVD) as methylation control

• Protein expression analysis:

  • Use FITC-conjugated FAM110C antibodies for detection by immunofluorescence or flow cytometry

  • Include methylated cell lines (e.g., MIAPaCa-2, JF-305) and unmethylated cell lines (e.g., Panc3.11, Panc10.05) as controls

  • Quantify expression levels using standardized image analysis or flow cytometry metrics

• Functional validation:

  • Treat methylated cell lines with 5-aza-2'-deoxycytidine (2 μM for 96h) to confirm methylation-dependent regulation

  • Perform CRISPR knockout of FAM110C to establish baseline for complete loss-of-function

  • Create stable FAM110C-expressing cell lines using lentiviral vectors for gain-of-function studies

• Clinical correlation:

  • Correlate methylation status and protein expression with patient demographics and outcomes

  • Use Kaplan-Meier survival curves with log-rank tests to assess prognostic value

  • Employ multivariate Cox regression analysis to identify independent prognostic factors

This integrated approach has revealed that in PDAC, FAM110C methylation is an independent predictor of poor survival (HR=0.544, p=0.016) , while in GBM, high protein expression correlates with worse outcomes (HR=1.55, p=0.03) , demonstrating the complementary value of combining methylation and protein analyses.

What are the technical considerations for using FAM110C-FITC antibodies in multiplex immunofluorescence assays?

Developing effective multiplex immunofluorescence assays incorporating FAM110C-FITC antibodies requires addressing several technical challenges:

Spectral considerations:

• FITC emits green fluorescence (emission ~519nm) that may overlap with other green-emitting fluorophores

• Optimal fluorophore combinations with FITC include DAPI (blue), TRITC/Cy3 (red), and Cy5 (far-red)

• When using confocal microscopy, utilize narrow bandpass filters and sequential scanning to minimize bleed-through

Panel design for cancer-specific FAM110C studies:

Staining protocol optimization:

• Test antibody combinations on single-stained controls to assess cross-reactivity

• Determine optimal sequence for multiple antibody applications (concurrent vs. sequential)

• Include appropriate blocking steps between antibody applications if using sequential staining

• Validate multiplex results against single-staining controls to confirm specificity

Image acquisition and analysis:

• Capture each fluorophore using appropriate filter sets with minimal overlap

• Employ spectral unmixing algorithms for closely overlapping fluorophores

• Use automated quantification methods for objective assessment of co-localization

• Normalize FAM110C intensity to account for cell-to-cell variability in expression

Successful multiplex assays provide contextual understanding of FAM110C's interactions with other proteins and pathways, enhancing its value as a biomarker in complex cancer biology studies.

How can I troubleshoot non-specific binding or high background when using FAM110C-FITC antibodies?

Troubleshooting non-specific binding and high background requires systematic evaluation of multiple parameters:

Common issues and solutions for FAM110C-FITC antibody staining:

FAM110C-specific troubleshooting guidance:

• Tissue-specific considerations:

  • For PDAC samples, check FAM110C methylation status as 72.89% show methylation

  • For GBM, verify IDH1 mutation status as expression varies significantly between wild-type and mutant

• Subcellular localization issues:

  • If observing only nuclear or only cytoplasmic staining, optimize permeabilization protocols

  • FAM110C should be detected in both compartments based on IHC data

• Scientific validation approaches:

  • Confirm antibody specificity with western blot

  • Validate expression changes with qRT-PCR

  • Use siRNA/shRNA knockdown to confirm specificity of staining

  • Compare results with published expression patterns in specific cancer types

Systematic troubleshooting not only improves experimental quality but can also reveal important biological insights about FAM110C regulation in different cancer contexts.

What are the emerging applications of FAM110C as a synthetic lethal marker in cancer therapy research?

Recent research has uncovered FAM110C's role in synthetic lethal interactions, opening new avenues for targeted cancer therapy:

Mechanism of synthetic lethality:

• In PDAC, FAM110C activates ATM and NHEJ signaling pathways by interacting with HMGB1

• Loss of FAM110C through methylation creates vulnerability to ATR/CHK1 inhibition

• This represents a classic synthetic lethal interaction where cells can survive either FAM110C loss OR ATR/CHK1 inhibition, but not both simultaneously

Experimental validation in PDAC:

• FAM110C-silenced cells showed significantly increased sensitivity to VE-822 (ATR inhibitor) and MK-8776 (CHK1 inhibitor)

• Half-inhibitory concentration (IC₅₀) analysis demonstrated reduced cell viability in FAM110C-negative cells treated with these inhibitors

• This synthetic lethal relationship was confirmed across multiple PDAC cell lines including MIAPaCa-2, JF-305, and Panc10.05

Potential therapeutic applications:

• Patient stratification: Screening for FAM110C methylation (72.89% of PDAC patients) could identify candidates for ATR/CHK1 inhibitor therapy

• Combination strategies: Demethylating agents plus ATR/CHK1 inhibitors might show synergistic effects

• Resistance mechanisms: FAM110C re-expression could contribute to resistance to ATR/CHK1 inhibitors

• Biomarker development: FAM110C methylation status could serve as companion diagnostic for ATR/CHK1 inhibitor trials

Emerging applications in GBM:

• Computational mapping analysis identified two potential drugs targeting FAM110C in GBM:

  • Felibinac (Mean connective score = −0.554; p=0.001)

  • Fludrocortisone (Mean connective score = −0.561; p=0.001)

• Both drugs showed dose-dependent inhibition of GBM cell proliferation by reversing FAM110C effects

These findings highlight FAM110C's emerging importance not only as a biomarker but also as a determinant of therapeutic response in precision oncology approaches.

How can quantitative flow cytometry with FAM110C-FITC antibodies provide insights into cell cycle regulation?

FAM110C's established role in G1/S arrest makes flow cytometric analysis particularly valuable for understanding its cell cycle regulatory functions:

Methodological approach for cell cycle analysis:

• Cell synchronization protocol:

  • Synchronize cells to G0/G1 phase by serum withdrawal for 12 hours

  • Re-enter cells into the cell cycle by adding serum (10% FBS) for 36 hours

  • Analyze at multiple timepoints to track cell cycle progression

• Multiparameter flow cytometry setup:

  • FAM110C-FITC antibody for protein detection

  • Propidium iodide or other DNA stain for cell cycle phase determination

  • Annexin V for apoptosis assessment if studying cell death

• Quantitative analysis framework:

  • Gate viable single cells based on scatter properties

  • Create bivariate plots of FAM110C expression versus DNA content

  • Analyze FAM110C expression levels across G0/G1, S, and G2/M populations

  • Compare expression patterns between control and experimental conditions

Data interpretation guidelines:

Advanced applications:

• Compare FAM110C expression dynamics between methylated vs. unmethylated cell lines

• Analyze the impact of 5-aza treatment on cell cycle distribution and FAM110C expression

• Evaluate the effects of ATR/CHK1 inhibitors on FAM110C-expressing versus FAM110C-silenced cells

• Correlate FAM110C expression with cell cycle checkpoint proteins (p53, p21, cyclins)

This approach has revealed that FAM110C inhibits PDAC cell growth by inducing G1/S arrest, while its silencing through methylation may contribute to dysregulated cell cycle control and cancer progression .

What considerations should guide the selection and validation of FAM110C antibodies for specific research applications?

Selecting and validating appropriate FAM110C antibodies requires careful consideration of multiple factors:

Selection criteria for FAM110C antibodies:

Comprehensive validation strategy:

• Expression verification:

  • Compare antibody staining with mRNA expression data

  • Verify expression patterns match known FAM110C distribution (nuclear and cytoplasmic)

• Specificity controls:

  • Use CRISPR knockout cell lines to confirm antibody specificity

  • Compare methylated cell lines (negative) with unmethylated cell lines (positive)

  • Test 5-aza-treated cells to confirm methylation-dependent expression

• Technical validation:

  • Perform western blot analysis to confirm detection of the expected ~36 kDa band

  • Validate subcellular localization with fractionation studies

  • Compare results from multiple antibodies targeting different epitopes

• Application-specific validation:

  • For flow cytometry: optimize fixation/permeabilization protocols for intracellular staining

  • For IHC/IF: test multiple antigen retrieval methods on FFPE tissues

  • For functional studies: confirm antibody doesn't interfere with protein interactions

• Disease-specific considerations:

  • For PDAC: validate correlation between methylation status and antibody staining

  • For GBM: confirm higher expression in wild-type versus IDH1-mutant samples

Thorough validation ensures reliable results and enables meaningful interpretation of FAM110C expression patterns across different experimental contexts and disease states.

How does FAM110C interact with DNA damage repair pathways in cancer, and how can FITC-conjugated antibodies help elucidate these mechanisms?

FAM110C has emerged as an important regulator of DNA damage repair (DDR) pathways with significant implications for cancer biology:

Key mechanistic findings:

• FAM110C activates ATM and Non-Homologous End Joining (NHEJ) signaling pathways

• This activation occurs through direct interaction with High Mobility Group Box 1 (HMGB1)

• Loss of FAM110C expression in PDAC sensitizes cells to ATR/CHK1 inhibitors

• This creates a synthetic lethal relationship between FAM110C silencing and ATR/CHK1 inhibition

Experimental approaches using FITC-conjugated antibodies:

• Co-localization studies:

  • Use FITC-conjugated FAM110C antibodies alongside differently labeled DDR proteins

  • Perform confocal microscopy to assess co-localization at DNA damage sites

  • Quantify spatial relationships between FAM110C and key DDR factors before and after DNA damage induction

• Proximity ligation assays:

  • Combine FAM110C-FITC antibodies with proximity ligation techniques

  • Visualize and quantify specific protein-protein interactions in situ

  • Assess dynamic changes in interaction frequency following DNA damage

• Chromatin immunoprecipitation:

  • Use FAM110C antibodies for ChIP experiments to identify genomic binding sites

  • Determine if FAM110C associates with chromatin at specific genomic regions

  • Correlate binding patterns with DNA damage response elements

• Live-cell imaging:

  • Develop strategies to use FITC-conjugated antibody fragments in live cells

  • Track FAM110C recruitment to DNA damage sites in real-time

  • Analyze kinetics of accumulation and dissociation

Research applications in cancer biology:

• Compare FAM110C recruitment patterns between normal and cancer cells

• Evaluate changes in FAM110C-DDR protein interactions in response to chemotherapy

• Assess how FAM110C methylation status affects DDR pathway activation

• Determine whether FAM110C expression predicts sensitivity to DNA-damaging agents

These approaches using FITC-conjugated FAM110C antibodies can help elucidate the mechanisms by which FAM110C contributes to genome stability and how its dysregulation influences cancer development and therapeutic response.

What emerging technologies can enhance the detection and functional analysis of FAM110C using fluorescently labeled antibodies?

Several cutting-edge technologies are advancing the capabilities for FAM110C detection and functional analysis:

Mass cytometry (CyTOF):

• Combines antibody specificity with mass spectrometry detection

• Enables simultaneous measurement of 40+ proteins including FAM110C

• Eliminates spectral overlap issues of fluorescent antibodies

• Requires metal-conjugated rather than FITC-conjugated antibodies

• Particularly valuable for comprehensive analysis of FAM110C in complex signaling networks

Super-resolution microscopy:

• Techniques like STORM, PALM, and STED bypass diffraction limit

• Achieves resolution of 20-50nm versus 200-300nm in conventional microscopy

• Allows precise localization of FAM110C within subcellular structures

• Can resolve FAM110C interactions with microtubules and other cytoskeletal elements

• Requires specialized FITC-conjugated antibodies optimized for super-resolution techniques

Single-cell omics integration:

• Combines antibody-based protein detection with transcriptomics/genomics

• CITE-seq allows simultaneous measurement of FAM110C protein and mRNA

• Correlates FAM110C protein expression with methylation status at single-cell level

• Particularly valuable for heterogeneous cancer samples to identify subpopulations

Spatial transcriptomics with protein detection:

• Integrates in situ RNA analysis with antibody-based protein detection

• Maps FAM110C expression patterns across tissue architecture

• Correlates spatial distribution with functional states in tumor microenvironment

• Technologies like 10x Visium with immunofluorescence can reveal tissue-level organization

Organoid and 3D culture systems with advanced imaging:

• Uses FITC-conjugated FAM110C antibodies in 3D cancer models

• Light-sheet microscopy allows deep tissue penetration with minimal photobleaching

• Captures FAM110C dynamics in physiologically relevant contexts

• Particularly valuable for studying FAM110C in cell migration and invasion in 3D

These technologies can provide unprecedented insights into FAM110C biology across scales from molecular interactions to tissue-level organization, advancing our understanding of its roles in cancer progression and potential as a therapeutic target.

How might FAM110C-FITC antibodies contribute to developing precision medicine approaches for cancer treatment?

FAM110C-FITC antibodies have significant potential to advance precision medicine approaches for cancer treatment:

Patient stratification applications:

Treatment monitoring applications:

Emerging precision applications:

• Liquid biopsy development:

  • Detection of FAM110C methylation in circulating tumor DNA

  • Monitoring dynamic changes in methylation patterns during treatment

  • Non-invasive assessment of tumor evolution and treatment response

• Targeted drug delivery:

  • FAM110C antibodies conjugated to nanoparticles or drug delivery systems

  • Selective targeting of FAM110C-expressing cells in GBM

  • Potential for antibody-drug conjugates targeting FAM110C-positive cells

• Combination therapy rationalization:

  • FAM110C status could guide optimal drug combinations

  • Example: In FAM110C-methylated PDAC, combining demethylating agents with ATR/CHK1 inhibitors

  • Simultaneous targeting of multiple pathways based on FAM110C-associated vulnerabilities

These precision medicine applications highlight how FAM110C-FITC antibodies extend beyond basic research tools to potentially actionable clinical applications that could improve cancer treatment outcomes.