FANCC Antibody

What is FANCC and why is it significant in biomedical research?

FANCC (Fanconi anemia complementation group C) is a critical protein involved in the Fanconi anemia (FA) pathway, which functions primarily in DNA damage repair, particularly in resolving interstrand cross-links (ICLs). FANCC is one of eight proteins that form the FA core complex, which activates FANCD2 and FANCI proteins to facilitate DNA repair. Approximately 80-90% of Fanconi anemia cases are due to variants in one of three genes: FANCA, FANCC, and FANCG .

Research significance:

• FANCC mutations are associated with bone marrow failure, developmental abnormalities, and predisposition to acute myeloid leukemia and other cancers

• FANCC is implicated in immune function, including B cell differentiation and antibody production

• Studies show FANCC interactions with proteins outside the traditional FA pathway, including UNC5A which is involved in neuronal development

Understanding FANCC through antibody-based detection provides insights into DNA repair mechanisms, cancer development, and potential therapeutic targets for FA patients.

What types of FANCC antibodies are available and how should researchers select appropriate antibodies?

Researchers have several options for FANCC antibodies with distinct characteristics:

Selection criteria should include:

• Experimental application (WB, IHC, IP, ICC)

• Species being studied (human, mouse, rat)

• Target epitope and antibody validation data

• Positive controls used in validation

• Storage conditions (typically in 50% Glycerol, 0.05% Azide, 1% BSA at -20°C)

For knockout studies or negative controls, researchers should consider using FANCC-deficient cell lines such as PD331, VU1131, or available FANCC knockout mice .

How can researchers validate FANCC antibody specificity for experimental use?

Comprehensive validation of FANCC antibodies should follow these methodological steps:

• Positive/negative control comparison: Use validated FANCC-proficient and FANCC-deficient cell lines

  • Example controls from literature: HEK293T (positive) vs. PD331 (FANCC-deficient)

  • VU1131 FANCC vs. S91 (FANCC-deficient)

• Verification of molecular weight: FANCC should be detected at approximately 63 kDa

• Mutant analysis: Test antibody against FANCC mutants like L554P and R548X

  • Data shows that the FANCC (541:558) antibody recognizes wild-type FANCC and barely detects FANCC-L554P, but does not recognize R548X mutant

• Cross-reactivity testing: If working with multiple species, verify reactivity as specified

  • Example: DF6632 antibody is reactive to human and mouse FANCC, with predicted reactivity to horse, rabbit, and dog

• Secondary antibody optimization: For Western blot, ECL Anti-Rabbit HRP secondary (1:5000 dilution) has been validated with primary FANCC antibodies at 1:1000-1:2000 dilution

A rigorous validation approach ensures experimental reliability and interpretable results.

What are the optimal conditions for Western blot detection of FANCC?

Optimized Western blot conditions for FANCC detection based on published protocols:

Sample preparation:

• Prepare whole cell lysates with 30 μg protein loading for optimal detection

• Use 6-8% SDS-PAGE gels for optimal resolution of FANCC (63 kDa)

Transfer conditions:

• Transblot at 25V for 13 minutes using Biorad Transfer buffer 1X

• Alternatively, standard overnight transfer at 30V has been successful

Antibody conditions:

• Primary antibody:

  • FANCC (541:558): 1:2000 dilution (0.5 μg/mL), 1-hour room temperature incubation

  • FANCC (41:56): 1:1000 dilution, overnight at 4°C incubation

• Secondary antibody:

  • Anti-rabbit HRP: 1:5000-1:10,000 dilution, 1-hour room temperature incubation

Detection:

• Chemiluminescence detection (e.g., Clarity Western ECL Substrate) provides optimal results

• Exposure time optimization is essential as FANCC expression varies by cell type

For researchers encountering detection issues, increasing antibody concentration or extending incubation time may improve signal, while maintaining FANCC specificity.

How can researchers effectively use FANCC antibodies for immunoprecipitation studies?

Immunoprecipitation (IP) with FANCC antibodies has been successfully used to study protein-protein interactions and post-translational modifications. The following protocol is based on published methodologies:

IP Protocol Optimization:

• Cell lysis: Use IP-compatible lysis buffer (typically containing 1% NP-40 or Triton X-100)

• Pre-clearing: Incubate lysate with protein A/G beads for 1 hour at 4°C

• Antibody binding: Incubate cleared lysate with FANCC antibody (2-5 μg per 1 mg protein)

  • Mouse monoclonal 8F3 has demonstrated efficacy for IP of FANCC from HeLa cell lysates

• Capture: Add protein A/G beads and incubate overnight at 4°C

• Washing: Perform 3-5 washes with cold IP buffer

• Elution: Use SDS loading buffer and heat at 95°C for 5 minutes

Experimental considerations:

• Co-IP experiments should include appropriate IgG controls

• For studying FANCC interactions within the FA core complex, consider mild detergent conditions

• When investigating DNA damage-induced interactions, treat cells with DNA cross-linking agents like MMC (mitomycin C) before lysis

Published studies have used FANCC antibodies for IP to demonstrate interactions with proteins both within and outside the FA pathway, offering insights into novel FANCC functions.

What methods are available to study FANCC expression in tissue samples?

Immunohistochemistry (IHC) and immunofluorescence approaches for FANCC detection in tissues require specific optimization:

IHC Protocol Guidelines:

• Fixation: 4% paraformaldehyde or 10% neutral buffered formalin

• Antigen retrieval: Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0)

• Blocking: 5% normal serum in PBS with 0.1% Triton X-100

• Primary antibody:

  • DF6632 has been validated for IHC applications

  • Recommended dilution: 1:100-1:200 for paraffin sections

  • Incubation: Overnight at 4°C

• Detection: DAB or fluorescence-based secondary antibody systems

Tissue expression patterns:
FANCC shows ubiquitous expression across tissues , but expression levels may vary. Higher expression has been noted in:

• Bone marrow hematopoietic cells

• Lymphoid tissues

• Proliferating epithelial cells

Experimental controls:

• Positive control: Normal human/mouse tissues with verified FANCC expression

• Negative control: FANCC-deficient tissues from knockout models or omission of primary antibody

• Specificity control: Validation with peptide competition assays

Researchers should note that FANCC subcellular localization can be both nuclear and cytoplasmic, with localization patterns potentially changing in response to DNA damage.

How can FANCC antibodies be utilized to investigate Fanconi anemia pathways?

FANCC antibodies provide powerful tools for investigating the FA pathway in both normal and disease contexts:

Core FA pathway investigation:

• FANCC monoubiquitination analysis:

  • Western blot to detect FANCC as part of the FA core complex

  • Co-IP to study FANCC interactions with other FA core proteins (FANCA, FANCB, etc.)

• FANCD2/FANCI activation monitoring:

  • Use FANCC antibodies in conjunction with FANCD2 antibodies to correlate FANCC expression with FANCD2 monoubiquitination

  • Studies show that Fancc−/−;Fancg−/− mice fail to monoubiquitinate FANCD2, confirming the essential role of FANCC in this process

• DNA damage response studies:

  • Treat cells with DNA cross-linking agents (e.g., MMC, cisplatin)

  • Use FANCC antibodies to track protein recruitment to damage sites

  • Immunofluorescence co-localization with γH2AX foci

Methodological advantages:

• Combinatorial approaches using multiple FA protein antibodies provide comprehensive pathway analysis

• Western blot and IP approaches allow quantification of protein levels and complex formation

• Immunofluorescence permits visualization of spatial and temporal dynamics

Such studies have revealed that FANCC and FANCG function in both overlapping and divergent molecular pathways, with combined inactivation leading to cooperative impairment of hematopoietic stem cell function .

What role do FANCC antibodies play in studying B cell function and immune response?

Recent research has revealed unexpected roles for FANCC in immune function, particularly in B cell development and antibody production:

Key experimental approaches:

• B cell differentiation analysis:

  • Flow cytometry with FANCC antibodies to correlate protein expression with B cell developmental stages

  • Studies show Fancc−/− B cells have impaired Antibody-Secreting-Cell (ASC) differentiation

• Antibody production assessment:

  • In vitro analysis of IgM and IgG production in FANCC-deficient B cells

  • Data shows Fancc−/− B cells have a specific defect in IgG2a class switching

• Wnt signaling investigation:

  • Western blot analysis using FANCC and β-catenin antibodies reveals hyper-active Wnt signaling in Fancc−/− B cells

  • This provides a potential mechanism for impaired ASC differentiation

Experimental evidence:
Fancc−/− mice show:

• Decreased proportion of CD138+B220dim/- cells (ASCs) after LPS stimulation

• Reduced serum IgM concentration compared to wild-type mice

• Lower levels of NP-specific IgG after immunization with NP-LPS

• Hyper-active Wnt signaling and accumulated β-catenin in B cells

These findings establish FANCC as a regulator of B cell function and identify the Wnt pathway as a potential therapeutic target for FA immune deficiency.

How can researchers effectively use FANCC antibodies in cancer research?

FANCC antibodies have important applications in cancer research, particularly for investigating DNA repair deficiencies and potential therapeutic vulnerabilities:

Research applications:

• Diagnostic/prognostic biomarker assessment:

  • IHC staining of tumor tissues to evaluate FANCC expression

  • Correlation of expression with clinical outcomes and treatment response

• DNA repair deficiency analysis:

  • Western blot to detect FANCC in various cancer cell lines

  • Identification of cancers with FA pathway defects that may be sensitive to specific therapies

• Drug response studies:

  • Use FANCC antibodies to monitor protein expression and localization before and after treatment

  • FA-deficient cancers show hypersensitivity to DNA cross-linking agents and PARP inhibitors

• Cancer model development:

  • FANCC antibodies are used to validate FANCC knockout or knockdown in cancer cell lines

  • The Fanconi Anemia Research Materials program provides FANCC-deficient cancer cell lines for research

Experimental significance:
FA deficiency contributes to genomic instability in various cancers. Studies using FANCC antibodies have shown that:

• FA patients have increased risk of developing acute myeloid leukemia (AML)

• FA patients also have elevated risk for tumors of the liver, gastrointestinal system, and head and neck squamous cell carcinoma (HNSCC)

• FANCC-deficient cell lines can be used to identify synthetic lethal interactions and new therapeutic targets

This research has significant translational implications for both FA patients and potentially for sporadic cancers with FA pathway defects.

What are common technical challenges with FANCC antibodies and how can they be resolved?

Researchers frequently encounter specific challenges when working with FANCC antibodies:

Methodological recommendations:

• When validating a new FANCC antibody, test multiple dilutions (1:500, 1:1000, 1:2000) to determine optimal signal-to-noise ratio

• Include both positive (FANCC-expressing) and negative (FANCC-deficient) controls in each experiment

• For challenging samples, consider membrane stripping and re-probing with a different FANCC antibody targeting a different epitope

These approaches have successfully resolved detection issues in published FANCC studies.

How should researchers interpret conflicting results between different FANCC antibodies?

When faced with discrepancies in results using different FANCC antibodies, researchers should follow this methodological framework:

Systematic analysis approach:

• Epitope mapping comparison:

  • FANCC (41:56) targets the N-terminal region

  • FANCC (541:558) targets a C-terminal region

  • Differential results may reflect epitope accessibility or post-translational modifications

• Antibody validation strength:

  • Review validation data for each antibody using knockout controls

  • The FANCC (541:558) antibody has documented specificity against mutant forms (recognizes wild-type and weakly detects L554P but not R548X)

• Functional domain considerations:

  • N-terminal domain (aa 1-306) is involved in FANCE binding

  • C-terminal domain contains nuclear localization signals

  • Different antibodies may detect functionally distinct FANCC populations

• Experimental context evaluation:

  • Cell/tissue type differences in FANCC expression or processing

  • Treatment conditions affecting protein conformation or interactions

  • Sample preparation methods impacting epitope exposure

When faced with discrepancies, researchers should report results from multiple antibodies and consider the biological significance of differences rather than dismissing them as technical artifacts.

What are the latest methodologies incorporating FANCC antibodies in cutting-edge research?

Emerging research approaches utilizing FANCC antibodies include:

Advanced methodological applications:

• Proximity ligation assays (PLA):

  • Combines FANCC antibodies with antibodies against potential interacting proteins

  • Allows visualization and quantification of protein-protein interactions in situ

  • Particularly valuable for studying FANCC interactions outside the FA core complex

• ChIP-seq and CUT&RUN applications:

  • Using FANCC antibodies to identify genomic binding sites

  • Helps elucidate potential transcriptional regulation roles beyond DNA repair

  • Relevant for understanding FANCC's role in gene expression regulation

• Mass spectrometry-based approaches:

  • Immunoprecipitation with FANCC antibodies followed by MS analysis

  • Identifies novel interacting partners and post-translational modifications

  • Creates comprehensive interaction networks

• Super-resolution microscopy:

  • Combines FANCC antibodies with techniques like STORM or PALM

  • Provides nanoscale resolution of FANCC localization and dynamics

  • Reveals previously undetectable spatial organization

• Organoid and patient-derived xenograft (PDX) models:

  • IHC with FANCC antibodies to characterize FA pathway status in 3D culture systems

  • Allows evaluation of FANCC expression in more physiologically relevant contexts

  • Facilitates personalized medicine approaches for FA patients

These emerging methodologies are expanding our understanding of FANCC beyond its classic role in the FA core complex, revealing novel functions in immune regulation, neuronal development, and other cellular processes.

What validated FANCC-deficient cell models are available for antibody validation?

Researchers seeking controls for FANCC antibody validation have access to several well-characterized models:

Human cell lines:

• PD331: FANCC-deficient patient-derived fibroblast line

• VU1131: FANCC-deficient fibroblast line, commonly used as negative control

• PD331/C: FANCC-complemented version of PD331, serves as positive control

Mouse models:

• Fancc−/− mice: Available knockout model with characterized hematopoietic and immune phenotypes

• Fancc−/−;Fancg−/− double knockout mice: Shows more severe phenotype than single knockouts

Specialized resources:

• Oregon Health & Science University (OHSU) in partnership with the Fanconi Cancer Foundation maintains the Fanconi Cell Line Repository

• This repository provides immortalized human FA fibroblast cell lines, including FANCC-deficient lines

• Isogenic cell pairs (FANCC-mutant and FANCC-complemented) are available

These validated models ensure proper controls for antibody specificity testing and functional studies. Researchers can access many of these resources through the Fanconi Anemia Research Materials program at OHSU.

How should researchers properly cite and acknowledge FANCC antibody resources?

When using FANCC antibodies from established repositories, proper citation is essential for resource sustainability:

Citation guidelines:

• Commercial antibodies:

  • Include catalog number, clone, and manufacturer

  • Example: "Anti-FANCC Antibody (clone 8F3, MABC524, Sigma-Aldrich)"

  • Include RRID (Research Resource Identifier) when available: "RRID:AB_2838594"

• Repository antibodies:

  • For Fanconi Anemia Research Materials antibodies, use: "We thank Fanconi Anemia Research Materials, funded by Fanconi Anemia Research Fund through a partnership with Oregon Health & Science University, for providing the antibodies to enable the research reported here."

  • Include specific antibody identifier (e.g., "FANCC (41:56), C3831")

• Publication citations:

  • Cite relevant methodology papers

  • For novel applications or validations, consider publishing antibody validation data

Data submission requirements:

• Users of Fanconi Anemia Research Materials antibodies must submit use-data back to the repository

• Contact repository managers (e.g., Leslie Wakefield, wakefiel@ohsu.edu) when ready to submit data

• Include both positive and negative results to benefit the research community

Proper citation and data feedback ensure continued availability of these valuable research tools and advance the collective knowledge about FANCC antibodies.