FAM110C Antibody, HRP conjugated

What is FAM110C and why is it significant in research applications?

FAM110C (Family with sequence similarity 110C) is a cell fate-related gene that functions as a tumor suppressor, particularly in pancreatic ductal adenocarcinoma (PDAC). The protein plays several critical roles in cellular function:

• Inhibits cell proliferation by inducing G1/S arrest

• Suppresses cell migration by inhibiting AKT signaling through interaction with the microtubule cytoskeleton

• Activates ATM and NHEJ (Non-Homologous End Joining) signaling pathways by interacting with HMGB1

• Functions in DNA damage repair mechanisms

How does HRP conjugation enhance antibody functionality in research assays?

Horseradish Peroxidase (HRP) conjugation significantly enhances antibody functionality through several mechanisms:

• Catalytic amplification: HRP catalyzes the oxidation of substrates, producing a detectable signal with high signal-to-noise ratio

• Versatile detection options: Compatible with chromogenic, fluorogenic, and chemiluminescent substrates for multiple detection modalities

• Direct detection workflow: Eliminates the need for secondary antibodies, simplifying experimental protocols

• Stable signal generation: Provides reliable and reproducible signal output across different applications

HRP-conjugated antibodies are particularly valuable in techniques requiring high sensitivity and specificity, including ELISA, Western blotting, and immunohistochemistry. For FAM110C detection specifically, HRP conjugation offers an efficient way to visualize this protein in complex samples where expression may vary due to methylation status or cancer progression .

What are the key considerations when designing experiments with HRP-conjugated FAM110C antibodies?

When designing experiments with HRP-conjugated FAM110C antibodies, researchers should consider several critical factors:

Sample preparation considerations:

• FAM110C is expressed in both nuclear and cytoplasmic compartments, requiring extraction methods that effectively solubilize proteins from both locations

• Include protease inhibitors to prevent degradation during sample preparation

• For tissue samples, consider fixation methods that preserve FAM110C epitopes while maintaining cellular architecture

Experimental controls:

• Positive controls: Include cell lines with known FAM110C expression (e.g., Panc3.11, Panc5.04, and Panc10.05)

• Negative controls: Use cell lines with methylated FAM110C promoters and minimal expression (e.g., MIAPaCa-2 and JF-305)

• Validation controls: Consider CRISPR-knockout cell lines or siRNA knockdown samples to confirm antibody specificity

Application-specific optimizations:

• For Western blotting: Optimize protein loading (15-30 μg total protein)

• For IHC: Determine optimal antibody dilution (typically 1:100-1:500)

• For ELISA: Establish appropriate blocking agents to minimize background

Researchers should also account for the expression variability of FAM110C across different tissue types and disease states, particularly in cancer samples where methylation-driven silencing may reduce protein levels .

How do direct HRP-conjugated primary antibodies compare with two-step detection systems?

Direct HRP-conjugated primary antibodies and traditional two-step detection systems each offer distinct advantages and limitations:

For FAM110C detection specifically, the choice between direct and indirect systems should be based on:

• Expected abundance of FAM110C in the sample (consider methylation status)

• Required sensitivity of the assay

• Time constraints of the experimental workflow

• Need for multiplexing with other targets

Research indicates that direct HRP-conjugated antibodies are suitable when signal amplification is not needed, while indirect detection with secondary antibodies may improve signal for low-abundance targets .

What optimization strategies improve signal-to-noise ratio when using HRP-conjugated FAM110C antibodies?

Optimizing signal-to-noise ratio for HRP-conjugated FAM110C antibodies requires a systematic approach:

Antibody dilution optimization:

• Perform titration experiments with serial dilutions (1:500, 1:1000, 1:2000, 1:5000)

• Identify the concentration that maximizes specific signal while minimizing background

• For Western blotting applications, optimize based on expected FAM110C expression levels in specific samples

Blocking optimization:

• Test different blocking agents (BSA, non-fat dry milk, commercial blocking buffers)

• Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

• For samples with high background, consider adding 0.1-0.3% Triton X-100 to blocking buffer

Washing protocol refinement:

• Increase wash buffer volume and number of washes (minimum 3-5 washes of 5-10 minutes each)

• Consider using higher concentrations of Tween-20 (0.05-0.1%) in wash buffers

• For problematic samples, introduce brief high-salt wash steps to reduce non-specific interactions

Substrate selection:

• For Western blotting: Match the chemiluminescent substrate sensitivity to expected FAM110C abundance

• For IHC applications: Optimize substrate development time to maximize signal before background appears

• For ELISA: Consider enhanced chemiluminescent substrates for detecting low-abundance FAM110C

When working with cancer samples where FAM110C may be silenced through promoter methylation (as seen in up to 72.89% of PDAC cases), these optimization steps become particularly crucial for detecting potentially low protein levels .

How can FAM110C HRP-conjugated antibodies be used to investigate epigenetic regulation in cancer?

FAM110C HRP-conjugated antibodies can be powerful tools for investigating epigenetic regulation in cancer through several methodological approaches:

Correlation of methylation status with protein expression:

• Perform Western blotting using HRP-conjugated FAM110C antibodies on samples with known methylation status

• Quantify FAM110C protein levels and correlate with methylation data from MSP or bisulfite sequencing

• Research has shown FAM110C methylation in 72.89% of PDAC samples, with expression inversely correlated to methylation state

Treatment effects of demethylating agents:

• Analyze FAM110C protein levels before and after treatment with 5-aza-2'-deoxycytidine (5-aza)

• Use HRP-conjugated antibodies to quantify the restoration of expression following demethylation

• Data indicates 5-aza treatment successfully restores FAM110C expression in methylated cell lines

Tissue microarray analysis:

• Apply HRP-conjugated FAM110C antibodies to tissue microarrays from different cancer stages

• Correlate expression patterns with progression from precursor lesions (IPMN/MCN) to invasive cancer

• Research shows FAM110C methylation increases from 41.18% in IPMN to 72.89% in PDAC

Prognostic assessment:

This integrated approach allows researchers to connect FAM110C's epigenetic regulation with its protein expression and functional consequences in cancer development and progression.

What are the methodological approaches for studying FAM110C's role in DNA damage repair (DDR) using HRP-conjugated antibodies?

To investigate FAM110C's role in DNA damage repair using HRP-conjugated antibodies, researchers can employ several sophisticated methodological approaches:

Co-immunoprecipitation coupled with HRP detection:

• Immunoprecipitate FAM110C or interacting DDR proteins (such as HMGB1)

• Perform Western blotting with HRP-conjugated antibodies to detect interaction partners

• Research has demonstrated that FAM110C activates ATM and NHEJ signaling by interacting with HMGB1

Time-course analysis following DNA damage induction:

• Treat cells with DNA-damaging agents (e.g., VE-822, MK-8776, cisplatin)

• Collect samples at defined time points (0, 1, 3, 6, 24 hours)

• Use HRP-conjugated FAM110C antibodies to track expression changes during DDR activation

Subcellular fractionation analysis:

• Separate nuclear and cytoplasmic fractions before and after DNA damage

• Analyze FAM110C localization and potential translocation between compartments

• HRP-conjugated antibodies can provide quantitative assessment of compartment-specific expression levels

Synthetic lethality evaluation:

• Generate FAM110C knockout cell lines using CRISPR-Cas9 (targeting exons 1 and 2)

• Treat with ATR/CHK1 inhibitors at varying concentrations

• Assess cell viability using standard assays (MTT)

• Compare IC50 values between FAM110C-expressing and FAM110C-null cells to quantify synthetic lethality

Research has shown that loss of FAM110C expression sensitizes PDAC cells to ATR inhibitor VE-822 and CHK1 inhibitor MK-8776, demonstrating synthetic lethality that could be exploited therapeutically .

How can multiplexed detection strategies incorporate FAM110C HRP-conjugated antibodies?

Incorporating FAM110C HRP-conjugated antibodies into multiplexed detection strategies requires careful consideration of compatibility and optimization:

Sequential multiplexing approaches:

• Complete detection of FAM110C using HRP-conjugated antibody first

• Quench HRP activity completely using hydrogen peroxide or other quenching agents

• Proceed with detection of additional targets using different enzyme conjugates (e.g., alkaline phosphatase)

• This approach allows visualization of multiple proteins within the same sample

Dual enzyme detection systems:

• Use HRP-conjugated FAM110C antibodies alongside alkaline phosphatase (AP) conjugated antibodies

• Develop with substrates producing contrasting colors (e.g., DAB for HRP producing brown, and Fast Red for AP producing red)

• This approach works well for IHC applications studying protein co-localization

Biotin-streptavidin amplification:

• Employ biotin-conjugated FAM110C antibodies followed by streptavidin-HRP

• This approach provides signal amplification for detecting low-abundance FAM110C

• Can be combined with direct HRP conjugates of other targets

• Biotin-streptavidin systems are highly sensitive and used in ELISA, Western blotting, and immunohistochemistry

Alternative fluorophore systems:

• Consider fluorophore-conjugated versions of FAM110C antibodies (FL-490, FL-550, FL-594, FL-650)

• These can be combined with HRP-based detection of other targets

• Particularly useful for co-localization studies with other proteins involved in DDR or cytoskeletal interactions

When designing multiplexed detection experiments, researchers should validate each antibody individually before combining detection systems, and carefully optimize blocking, washing, and development steps to prevent cross-reactivity or interference between detection systems.

How can researchers validate the specificity of FAM110C HRP-conjugated antibodies?

Validating the specificity of FAM110C HRP-conjugated antibodies requires a multi-faceted approach:

Genetic manipulation controls:

• Generate FAM110C knockout cells using CRISPR-Cas9 technology (targeting exons 1 and 2)

• Create siRNA knockdown samples with reduced FAM110C expression

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

• Specific antibodies should show significantly reduced or absent signal in knockout samples

Expression correlation with methylation status:

• Test antibody in cell lines with known FAM110C methylation patterns

• Compare signal between unmethylated (e.g., Panc3.11, Panc5.04, Panc10.05) and methylated (e.g., MIAPaCa-2, JF-305) cell lines

• Specific antibodies should show correlation between signal intensity and methylation status

Ectopic expression validation:

• Generate stable cell lines overexpressing FAM110C using lentiviral vectors

• Confirm increased signal in overexpression models compared to controls

• Research protocols for stable expression systems are available using pCDH-CMV-MCS-puro plasmid constructs

Peptide competition assay:

• Pre-incubate antibody with the immunizing peptide (if available)

• Compare signal between blocked and unblocked antibody

• Specific signal should be significantly reduced or eliminated by peptide competition

Western blot validation:

• Confirm detection of a single band at the expected molecular weight

• Examine band intensity changes following 5-aza treatment in methylated cell lines

• Specific antibodies should show increased signal following demethylation treatment

This comprehensive validation approach ensures that signals detected using FAM110C HRP-conjugated antibodies accurately represent the target protein's presence and abundance.

What are common pitfalls when working with HRP-conjugated antibodies and how can they be addressed?

When working with HRP-conjugated antibodies, researchers may encounter several common challenges:

High background signal:

• Cause: Insufficient blocking, non-specific binding, or excessive antibody concentration

• Solution: Optimize blocking time and agent (BSA, non-fat milk, commercial blockers), increase wash steps, and perform antibody titration to determine optimal concentration

Weak or absent signal:

• Cause: Low target expression, epitope masking during fixation, or HRP inactivation

• Solution: For FAM110C specifically, consider treating cells with 5-aza to restore expression in methylated samples , optimize sample preparation, and ensure proper storage of HRP conjugates (4°C, avoid repeated freeze-thaw)

Non-specific bands in Western blotting:

• Cause: Cross-reactivity with related proteins or degradation products

• Solution: Increase blocking stringency, use monoclonal antibodies when available, and validate with appropriate positive/negative controls

Signal variability between replicates:

• Cause: Inconsistent sample loading, transfer efficiency variations, or detection system fluctuations

• Solution: Normalize to housekeeping proteins (e.g., GAPDH), standardize protein quantification methods, and use automated systems where possible

Excessive signal development in IHC applications:

• Cause: Endogenous peroxidase activity or overly sensitive substrate

• Solution: Include hydrogen peroxide quenching step, optimize substrate development time, and dilute substrate if necessary

Storage-related decline in performance:

• Cause: HRP degradation during storage

• Solution: Store at 4°C with proper preservatives, avoid repeated freeze-thaw cycles, and aliquot stock antibody to minimize exposure

For FAM110C specifically, the variable expression due to methylation status in different cancer samples requires careful optimization of detection sensitivity and appropriate positive controls .

How does FAM110C expression variability across tissue types impact antibody detection strategies?

FAM110C expression variability across tissue types significantly impacts antibody detection strategies, requiring tailored approaches:

Methylation-driven expression differences:

• FAM110C methylation increases with cancer progression: 41.18% in IPMN, 46.67% in MCN, and 72.89% in PDAC

• Adjust detection sensitivity based on expected methylation status in specific tissue types

• For highly methylated samples, consider signal amplification methods or longer exposure times

Subcellular localization considerations:

• FAM110C localizes to both nuclear and cytoplasmic compartments

• Ensure sample preparation methods effectively extract protein from all relevant cellular compartments

• For tissue sections, optimize antigen retrieval protocols to expose epitopes in all cellular locations

Tumor microenvironment effects:

• Expression may vary based on tumor microenvironment factors

• For heterogeneous samples, consider analyzing multiple regions

• In tissue microarrays, include multiple cores per sample to account for intratumoral heterogeneity

Detection strategy adaptations:

• For tissues with low expression: Increase antibody concentration, extend incubation time, or use more sensitive substrates

• For tissues with high expression: Dilute antibody appropriately to prevent signal saturation

• For comparative studies across different tissue types: Implement strict standardization of detection protocols

Quantification approaches:

• Use internal controls for normalization when comparing different tissue types

• Consider digital image analysis for objective quantification

• For Western blotting, normalize to housekeeping proteins that show stable expression across the tissues being compared

Understanding FAM110C's expression pattern in the specific tissue or disease context being studied is essential for designing appropriate detection strategies and correctly interpreting results, particularly in cancer research where FAM110C's prognostic significance has been established .

What emerging applications are being developed for FAM110C HRP-conjugated antibodies?

Emerging applications for FAM110C HRP-conjugated antibodies are expanding research capabilities in several directions:

Liquid biopsy development:

• Detection of FAM110C in circulating tumor cells or exosomes

• Potential use as a minimally invasive diagnostic tool for pancreatic cancer

• HRP-conjugated antibodies could offer the sensitivity needed for detecting low-abundance markers in complex biological fluids

Therapeutic response monitoring:

• Assessment of FAM110C expression changes during treatment with epigenetic modifiers

• Correlation with response to ATR/CHK1 inhibitors in synthetic lethal approaches

• HRP-based detection systems provide quantitative data suitable for longitudinal studies

Multiplex diagnostic panels:

• Integration of FAM110C with other methylation-sensitive markers

• Development of comprehensive prognostic panels for pancreatic cancer

• HRP-conjugated antibody arrays could enable simultaneous detection of multiple biomarkers

High-throughput screening applications:

• Screening for compounds that modulate FAM110C expression or function

• Identification of new synthetic lethal interactions

• HRP-based detection offers the throughput and sensitivity needed for large-scale screening efforts

Single-cell analysis techniques:

• Adaptation of HRP-conjugated antibodies for single-cell protein analysis

• Investigation of FAM110C heterogeneity within tumor populations

• Emerging technologies combining microfluidics with enzyme-based detection could enable these applications

As research into FAM110C's role in cancer biology and DNA damage repair continues to evolve, these emerging applications will likely expand to address new questions and clinical needs.

What future developments might improve HRP-conjugated antibody technology for research applications?

Future developments in HRP-conjugated antibody technology are likely to address current limitations and expand capabilities:

Enhanced enzyme variants:

• Engineered HRP variants with improved stability and catalytic efficiency

• Temperature-resistant forms for applications requiring higher incubation temperatures

• pH-tolerant variants for compatibility with a wider range of buffer conditions

Site-specific conjugation methods:

• Directed conjugation techniques that preserve antibody binding affinity

• Controlled enzyme-to-antibody ratios for more consistent performance

• These advances would improve batch-to-batch reproducibility and sensitivity

Multiplexing capabilities:

• Development of orthogonal substrate systems allowing multiple HRP-conjugated antibodies to be used simultaneously

• HRP variants engineered to work with specific substrates without cross-reactivity

• This would enable true multiplex detection using a single enzyme type

Smart detection systems:

• Integration with digital imaging platforms for automated quantification

• Machine learning algorithms for improved signal-to-noise discrimination

• These systems would enhance objectivity and reduce inter-observer variability

Nanobody and fragment-based conjugates:

• Smaller binding molecules conjugated to HRP for improved tissue penetration

• Reduced background through elimination of Fc-mediated interactions

• Particularly valuable for IHC applications in dense tissues

Reversible detection systems:

• Development of substrate systems that allow signal erasure and reprobing

• Would enable sequential detection of multiple targets on the same sample

• Particularly valuable for limited or precious samples