FAM110C Antibody, Biotin conjugated

What is FAM110C and why is it an important protein to study?

FAM110C (Family with sequence similarity 110 member C) is a protein that plays multiple critical roles in cellular function. It is primarily involved in:

• Microtubule organization during both interphase and mitosis

• Cell spreading and migration of epithelial cells, potentially through the AKT1 signaling pathway

• DNA damage repair mechanisms by activating ATM and NHEJ signaling pathways through interaction with HMGB1

The protein is detected in multiple tissues including stomach, thyroid, trachea, adrenal gland and testis, with lower expression levels in prostate, ovary, intestine, colon, spinal cord and lymph node .

How does biotin conjugation enhance the functionality of FAM110C antibodies?

Biotin conjugation provides several methodological advantages for FAM110C antibody applications:

• Enhanced detection sensitivity: The strong binding affinity between biotin and streptavidin (Kd ≈ 10^-15 M) creates a robust detection system that amplifies signals in assays like ELISA, Western blot, and immunohistochemistry .

• Versatile detection options: Biotin-conjugated antibodies can be detected using various streptavidin-conjugated reporter molecules (fluorophores, enzymes, quantum dots), allowing experimental flexibility .

• Multiplexing capability: When used alongside other primary antibodies with different conjugates, biotin-conjugated FAM110C antibodies enable simultaneous detection of multiple targets.

• Reduced cross-reactivity: The biotinylation process, when properly controlled, can minimize non-specific binding while maintaining the antibody's affinity for FAM110C .

The standard approach involves a two-step detection where the biotin-conjugated FAM110C antibody binds to the target protein, followed by detection using a streptavidin-reporter conjugate. This methodology significantly improves signal-to-noise ratios compared to direct detection methods .

What are the optimal applications for biotin-conjugated FAM110C antibodies?

Biotin-conjugated FAM110C antibodies are particularly useful in the following research applications:

Primary Applications:

• ELISA: For quantitative detection of FAM110C in complex samples. Biotin conjugation allows for sensitive detection using streptavidin-HRP systems .

• Immunohistochemistry (IHC): To visualize the tissue distribution and subcellular localization of FAM110C .

• Western Blot: For detection of denatured FAM110C protein in cell or tissue lysates .

• Immunocytochemistry: To study subcellular localization, particularly in relation to microtubules, centrosomes, and spindle poles during mitosis .

Advanced Applications:

• Protein capture with magnetic beads: Biotin-conjugated antibodies can be immobilized on streptavidin-coated magnetic beads for protein isolation and purification .

• Label-free and real-time binding assays: Compatible with Bio-Layer Interferometry (BLI) and Surface Plasmon Resonance (SPR) when immobilized on streptavidin-linked biosensors .

• In situ hybridization: For co-localization studies of FAM110C mRNA and protein expression patterns .

The choice of application should be guided by experimental objectives and the validated performance characteristics of the specific antibody .

What protocol modifications are necessary when using biotin-conjugated FAM110C antibodies in Western blot analysis?

When using biotin-conjugated FAM110C antibodies for Western blot analysis, several methodological adaptations are recommended:

Standard Protocol Modifications:

• Blocking step: Use biotin-free blocking solutions to prevent interference with the biotin-streptavidin interaction. Avoid milk-based blockers which contain endogenous biotin; instead, use 3-5% BSA in TBST or commercial biotin-free blockers .

• Detection system: Replace the typical secondary antibody step with a streptavidin-HRP or streptavidin-AP (alkaline phosphatase) conjugate at a dilution of 1:1000 to 1:5000 .

• Washing stringency: Include additional washing steps (5-6 washes of 5 minutes each) to reduce background signal, which is particularly important with biotin detection systems .

• Antibody dilution: Biotin-conjugated antibodies typically require higher dilutions than unconjugated ones. Start with a 1:500 dilution and optimize as needed .

• Exposure time: Shorter exposure times are often sufficient due to the signal amplification provided by the biotin-streptavidin system .

Troubleshooting Guidelines:

• If high background occurs, pre-clear samples with streptavidin beads to remove endogenous biotin.

• If signal is weak, ensure the biotin conjugate is fresh, as biotin activity can diminish over time with repeated freeze-thaw cycles.

• For multiplex detection, use different visualization methods for other proteins of interest to avoid detection conflicts .

How can FAM110C antibodies be used to investigate microtubule organization and spindle formation?

FAM110C has been shown to play a critical role in mitotic spindle formation through its interaction with both microtubules and actin. Biotin-conjugated FAM110C antibodies are valuable tools for examining these processes through the following methodological approaches:

Immunofluorescence Co-localization Studies:

• Triple staining protocol: Combine biotin-conjugated FAM110C antibody with fluorescently-labeled α-tubulin and phalloidin (for F-actin) to visualize the spatial relationship between FAM110C, microtubules, and actin filaments during different cell cycle phases .

• Fixation considerations: Use paraformaldehyde fixation (4%) for 15 minutes at room temperature to preserve cytoskeletal structures. Avoid methanol fixation which can disrupt actin filaments .

• Detection system: Use streptavidin conjugated to a spectrally distinct fluorophore (e.g., Alexa Fluor 647) for FAM110C detection, combined with directly labeled antibodies or probes for other markers .

Advanced Imaging Approaches:

• Live cell imaging: Transfect cells with GFP-FAM110C and mCherry-tubulin constructs, then perform time-lapse confocal microscopy to monitor dynamic interactions during spindle formation .

• Super-resolution microscopy: STORM or STED microscopy using biotin-conjugated FAM110C antibodies with appropriate streptavidin-fluorophore conjugates can reveal nanoscale organization of FAM110C in relation to spindle components .

• Proximity ligation assay: Combine biotin-conjugated FAM110C antibody with antibodies against potential interaction partners (e.g., HMGB1) to visualize and quantify protein-protein interactions in situ .

Research has demonstrated that FAM110C promotes interaction between kinetochore microtubules and spindle actin, with wild-type FAM110C efficiently promoting crosslinking of F-actin to immobilized microtubules in vitro . These assays provide important insights into FAM110C's role in spindle formation and chromosome segregation.

What are the considerations for using biotin-conjugated FAM110C antibodies in studies of cancer progression?

Recent research has identified FAM110C methylation as a potential diagnostic and prognostic marker in pancreatic cancer. When using biotin-conjugated FAM110C antibodies in cancer research, consider the following methodological aspects:

Experimental Design Considerations:

• Control selection: Include appropriate controls based on FAM110C methylation status. For instance, use cell lines with known FAM110C expression levels: Panc10.05 (unmethylated/high expression), PATU-8988T (partially methylated/moderate expression), and MIAPaCa-2 (fully methylated/no expression) .

• Tissue-specific expression: FAM110C expression varies across tissues. Consider this heterogeneity when selecting normal controls for comparison with tumor samples .

• Co-detection with methylation markers: When studying FAM110C in cancer progression, combine protein detection using biotin-conjugated antibodies with DNA methylation analysis for comprehensive characterization .

Methodological Approaches:

• Tissue microarray (TMA) analysis: For high-throughput screening of FAM110C expression across multiple tumor samples:

  • Use biotin-conjugated FAM110C antibody at 1:100-1:200 dilution

  • Employ streptavidin-HRP detection system

  • Score based on staining intensity and percentage of positive cells

• Correlation with clinical outcomes: Statistical analysis methods should include:

  • Kaplan-Meier survival analysis with log-rank testing

  • Cox regression analysis for multivariate analysis

  • Chi-square tests for association with clinicopathological parameters

• Functional studies: To investigate FAM110C's tumor suppressor role:

  • Use stable cell lines with FAM110C re-expression in methylated cell lines

  • Monitor effects on cell cycle (particularly G1/S transition), apoptosis, and proliferation

  • Assess DNA damage repair capacity through comet assays or γH2AX foci formation

How can I validate the specificity of biotin-conjugated FAM110C antibodies?

Validating antibody specificity is crucial for generating reliable research data. For biotin-conjugated FAM110C antibodies, employ the following comprehensive validation strategy:

Multi-level Validation Approach:

• Genetic knockout/knockdown controls:

  • Use CRISPR/Cas9-mediated knockout of FAM110C as the gold standard negative control

  • siRNA knockdown of FAM110C can serve as an alternative control

  • Compare antibody staining patterns between wild-type and knockout/knockdown samples

• Overexpression controls:

  • Use cells transfected with an FAM110C expression vector as a positive control

  • Include both wild-type FAM110C and tagged versions (e.g., FLAG-tagged or GFP-tagged)

  • Confirm co-localization of the biotin-conjugated antibody signal with the tag-specific antibody signal

• Peptide competition assay:

  • Pre-incubate the biotin-conjugated antibody with the immunizing peptide or recombinant FAM110C protein

  • Compare staining with and without peptide competition

  • Specific staining should be blocked by the competing peptide

Advanced Validation Techniques:

• Mass spectrometry validation:

  • Perform immunoprecipitation using the FAM110C antibody

  • Analyze the precipitated proteins by mass spectrometry

  • Confirm the presence of FAM110C peptides in the precipitated fraction

• Cell line panel analysis:

  • Test the antibody across a panel of cell lines with varying FAM110C expression levels

  • Correlate antibody signal intensity with mRNA levels measured by RT-PCR

  • Include cell lines known to have FAM110C methylation (e.g., MIAPaCa-2, JF-305) as negative controls

• Domain-specific validation:

  • Test the antibody against mutant FAM110C proteins with specific domain deletions

  • Key domains to consider include the actin-binding domain (aa40-61) and tubulin-binding domain (aa188-221)

  • This approach can also provide information about epitope location

Remember that proper validation ensures experimental reproducibility and the generation of reliable scientific data.

What are the technical challenges in detecting endogenous FAM110C in different cellular compartments?

Detecting endogenous FAM110C presents several technical challenges due to its dynamic subcellular localization and variable expression levels. Here are methodological approaches to address these challenges:

Challenges and Solutions:

• Dynamic localization during cell cycle:

  • FAM110C localizes to different cellular compartments depending on cell cycle phase (cytoplasm, cytoskeleton, centrosome, spindle poles, nucleus)

  • Solution: Synchronize cells at specific cell cycle stages using thymidine block (S-phase), nocodazole (G2/M), or serum starvation (G0/G1)

  • Co-stain with cell cycle markers (e.g., cyclin B1 for G2/M, Ki-67 for proliferating cells)

• Low endogenous expression levels:

  • FAM110C expression can be low or variable across cell types and tissues

  • Solution: Use signal amplification methods such as tyramide signal amplification (TSA) with biotin-conjugated antibodies

  • For Western blotting, enrich samples through immunoprecipitation before analysis

• Methylation-dependent expression:

  • FAM110C expression is regulated by promoter methylation, particularly in cancer cells

  • Solution: Treat cells with DNA methyltransferase inhibitors (e.g., 5-azacytidine) to restore expression in methylated cell lines

  • Include both methylated and unmethylated cell lines as controls

Optimization Protocols:

• Subcellular fractionation protocol:

  • Separate nuclear, cytoplasmic, and cytoskeletal fractions using differential centrifugation

  • Verify fraction purity with compartment-specific markers (e.g., lamin B1 for nucleus, GAPDH for cytoplasm)

  • Analyze FAM110C distribution across fractions by Western blot

• High-resolution imaging approach:

  • Use confocal microscopy with optimized optical sectioning (0.16 μm Z-stack intervals)

  • For centrosome/spindle pole localization, center ROIs (6 μm diameter) on γ-tubulin-positive structures

  • Quantify mean fluorescence intensity in defined compartments across multiple cells

• Extraction-resistant immunofluorescence:

  • Pre-extract cells with 0.5% Triton X-100 before fixation to remove soluble proteins

  • This approach enhances detection of cytoskeleton-associated FAM110C

  • Follow with standard immunofluorescence using biotin-conjugated antibody and streptavidin-fluorophore detection

Proper subcellular localization analysis is critical for understanding FAM110C function, as its roles in microtubule organization, spindle formation, and cell migration depend on its specific localization patterns.

How can I use biotin-conjugated FAM110C antibodies in multiplex immunoassays?

Multiplexing allows simultaneous detection of multiple targets in a single sample, providing valuable insights into protein co-expression and interaction networks. Here's how to effectively incorporate biotin-conjugated FAM110C antibodies into multiplex immunoassays:

Multiplex Immunofluorescence Protocol:

• Antibody panel design:

  • Select antibodies raised in different host species to avoid cross-reactivity

  • Combine biotin-conjugated FAM110C antibody with directly labeled antibodies against proteins of interest

  • For studies of FAM110C's role in spindle formation, include antibodies against α-tubulin, γ-tubulin, and actin markers

• Sequential staining approach:

  • Apply primary antibodies sequentially with stripping or blocking steps between rounds

  • Use the biotin-conjugated FAM110C antibody in the final round to prevent blocking of biotin binding sites

  • Visualize with spectrally distinct fluorophore-conjugated streptavidin

• Signal separation optimization:

  • Use narrow bandpass filter sets to minimize spectral overlap

  • Apply spectral unmixing algorithms during image analysis

  • Include single-stained controls for each fluorophore to generate spectral signatures

Flow Cytometry Multiplexing:

• Multi-parameter flow cytometry:

  • Combine biotin-conjugated FAM110C antibody with antibodies against cell cycle markers (e.g., propidium iodide for DNA content)

  • Use streptavidin conjugated to allophycocyanin (APC) or brilliant violet dyes for detection

  • Apply compensation controls to correct for spectral overlap

• Cell sorting protocol:

  • Sort cells based on FAM110C expression levels using the biotin-streptavidin detection system

  • Collect FAM110C-high and FAM110C-low populations for downstream analysis

  • Verify sorting purity by reanalysis of sorted populations

Multiplex Protein-Protein Interaction Analysis:

• Co-immunoprecipitation with biotinylated antibodies:

  • Use biotin-conjugated FAM110C antibody for immunoprecipitation

  • Capture complexes with streptavidin-coated magnetic beads

  • Identify interaction partners by Western blot or mass spectrometry

  • This approach has revealed FAM110C interactions with HMGB1, affecting DNA damage repair pathways

This multiplexing strategy allows comprehensive characterization of FAM110C's dynamic interactions and functional relationships in various cellular contexts.

What are the experimental considerations for using biotin-conjugated FAM110C antibodies in studying DNA damage repair mechanisms?

Recent research has identified FAM110C as a player in DNA damage repair through activation of ATM and NHEJ signaling pathways. When using biotin-conjugated FAM110C antibodies to study these mechanisms, consider the following methodological approaches:

Experimental Design for DNA Damage Studies:

• DNA damage induction protocols:

  • Use γ-irradiation (2-10 Gy) for double-strand breaks

  • Alternative agents: etoposide (topoisomerase II inhibitor) or hydroxyurea (replication stress)

  • Include time course analysis (0-24h post-damage) to track repair kinetics

• FAM110C and repair factor co-localization:

  • Combine biotin-conjugated FAM110C antibody with antibodies against DNA damage markers (γH2AX, 53BP1)

  • Use streptavidin-conjugated fluorophores spectrally distinct from repair factor labels

  • Quantify co-localization using Pearson's correlation coefficient or Manders' overlap coefficient

• Functional rescue experiments:

  • In FAM110C-depleted cells, re-express wild-type or mutant FAM110C lacking specific domains

  • Compare DNA repair efficiency between conditions

  • Particularly focus on the effect of the HMGB1-interaction domain on repair function

Advanced Methodological Approaches:

• ChIP-seq analysis protocol:

  • Use biotin-conjugated FAM110C antibody for chromatin immunoprecipitation

  • Sequence FAM110C-associated DNA to identify genomic binding sites

  • Compare binding patterns before and after DNA damage induction

• Synthetic lethality screening:

  • Combine FAM110C silencing with inhibitors of DNA repair pathways (ATR inhibitor VE-822, CHK1 inhibitor MK-8776)

  • Measure cell viability using MTT assay or colony formation assays

  • Calculate IC50 values to quantify synthetic lethal interactions

• Live-cell imaging of repair dynamics:

  • Use GFP-FAM110C expression constructs in cells with fluorescently tagged repair factors

  • Track recruitment kinetics to damage sites induced by laser microirradiation

  • Calculate recruitment rates and residence times at damage sites

Research has demonstrated that loss of FAM110C expression sensitizes pancreatic cancer cells to ATR/CHK1 inhibitors, with FAM110C methylation serving as a potential predictive biomarker for therapy response . This highlights the clinical relevance of understanding FAM110C's role in DNA repair mechanisms.

Data Table 1: FAM110C Expression and Methylation Status in Cell Lines

Data derived from methylation-specific PCR (MSP) and RT-PCR analysis

Data Table 2: Performance Characteristics of FAM110C Antibody Detection Methods