FANCA Antibody

What is FANCA protein and why are antibodies against it important for research?

FANCA (also known as FAA, FACA, or FANCH) is a DNA repair protein of approximately 162-163 kDa that shows widespread expression. It functions as a critical component in DNA repair mechanisms, specifically involved in interstrand DNA cross-link repair and maintenance of normal chromosome stability . The protein may operate in post-replication repair or cell cycle checkpoint functions .

FANCA antibodies are essential tools for studying Fanconi anemia (FA), a genetic disorder characterized by congenital abnormalities, bone marrow failure, and cancer predisposition . These antibodies enable detection of FANCA expression, localization, and interactions with other proteins in the FA pathway, making them crucial for understanding disease mechanisms and developing potential treatments.

Optimal dilutions vary by antibody and application:

It's important to note that these are general recommendations, and each laboratory should determine optimal dilutions for their specific experimental conditions .

What controls should be included when using FANCA antibodies?

Proper experimental controls are essential:

• Positive controls: Cell lines known to express FANCA, such as HeLa, HEK-293, Jurkat, MOLT-4, or A431 cells

• Negative controls: FANCA-deficient cell lines (e.g., 6914 pMMP-Empty) or FANCA knockout cell lines generated through CRISPR/Cas9

• Loading controls: GAPDH or other housekeeping proteins to normalize for total protein loading

• Secondary antibody-only controls: To assess non-specific binding

For validation studies, comparing antibody reactivity between wild-type and FANCA knockout samples provides the most definitive evidence of specificity .

How do polyclonal and monoclonal FANCA antibodies compare for research applications?

When choosing between antibody types, consider whether your experiment requires detection of specific modified forms of FANCA or if consistency across experiments is more critical. For detecting phosphorylated FANCA, which is important for its activation , antibodies recognizing specific phosphorylation sites would be preferable.

How can I troubleshoot weak or absent FANCA antibody signals in Western blot?

When facing challenges with FANCA detection:

• Protein extraction method: FANCA is a large protein (163 kDa) that may require specific lysis conditions. Multiple protocols show success with:

  • 2% SDS lysis buffer with 10% beta-mercaptoethanol

  • RIPA buffer supplemented with protease inhibitors

• Gel selection: Use gradient gels (3-8% Tris-Acetate or 4-12% Bis-Tris) for better resolution of high molecular weight proteins

• Transfer conditions:

  • Extended transfer times (60+ minutes) or higher voltage

  • Semi-dry transfer at 25V for 1 hour has shown success

• Antibody incubation:

  • Overnight primary antibody incubation at 4°C often yields better results than shorter incubations

  • Using 5% non-fat dry milk in TBST as blocking/dilution buffer

• Detection system:

  • Enhanced chemiluminescence substrate for higher sensitivity

  • Consider fluorescent secondary antibodies for quantitative analysis

• Sample preparation:

  • Avoid repeated freeze-thaw cycles of lysates

  • Include protease inhibitors in all buffers

If signal remains weak despite these optimizations, consider using a different FANCA antibody targeting a different epitope or concentrating your protein sample.

What is the significance of FANCA nuclear localization and foci formation in research applications?

FANCA normally relocates to the nucleus where it participates in the FA core complex formation and DNA repair . This nuclear localization and subsequent formation of nuclear foci after DNA damage are critical functional readouts in FA research:

• Nuclear localization:

  • FANCA contains a nuclear localization signal (NLS) at amino acids 18-34

  • Some missense mutations prevent proper nuclear localization of FANCA protein

  • Immunofluorescence studies can assess whether mutant FANCA proteins properly relocate to the nucleus

• Foci formation:

  • After DNA damage (e.g., from mitomycin C, hydroxyurea, or aphidicolin treatment), FANCA forms discrete nuclear foci

  • These foci represent sites of DNA repair complex assembly

  • Quantification of FANCA foci can serve as a functional assay for FA pathway activation

For immunofluorescence detection of FANCA foci:

• Treat cells with DNA crosslinking agents (e.g., 2mM hydroxyurea for 24 hours)

• Fix cells with 4% formaldehyde after hypotonic treatment

• Use FANCA antibodies at 1:500 dilution with appropriate fluorescent secondary antibodies

• Co-staining with γ-H2AX can confirm DNA damage sites

This approach allows researchers to assess the functional consequences of FANCA mutations or the effects of therapeutic interventions on the FA pathway.

How can FANCA antibodies be used to study interactions with other FA proteins?

FANCA forms complexes with other FA proteins, particularly FANCG, and participates in an eight-subunit Fanconi anemia core complex that ubiquitinates FANCD2 and FANCI . Studying these interactions provides insights into FA pathway regulation:

• Co-immunoprecipitation (Co-IP):

  • Use anti-FANCA antibodies to pull down FANCA protein complexes

  • Western blot with antibodies against other FA proteins to detect interactions

  • Example protocol: Immunoprecipitate FANCA from 1mg of cell lysate using antibodies at 1:30 dilution

• Proximity ligation assay (PLA):

  • Detects protein-protein interactions in situ with high sensitivity

  • Requires antibodies from different species for FANCA and interacting partners

  • Provides spatial information about where interactions occur within cells

• Chromatin immunoprecipitation (ChIP):

  • Can reveal FANCA association with specific DNA regions

  • Useful for studying FANCA recruitment to damaged DNA

• Sequential IP:

  • First IP with FANCA antibody followed by a second IP with antibody against potential interactor

  • Helps identify specific subcomplexes within larger protein assemblies

When designing interaction studies, consider that some antibody epitopes may be masked when FANCA is in protein complexes. Using antibodies targeting different regions of FANCA may yield complementary information.

What methods are recommended for validating FANCA antibody specificity?

Thorough validation is crucial for ensuring reliable results with FANCA antibodies:

• Genetic validation:

  • Compare antibody reactivity in wild-type versus FANCA knockout/knockdown cells

  • Example: "ab201457 was shown to specifically react with FANCA/FAA when FANCA/FAA knockout samples were used"

  • siRNA knockdown can also provide validation

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide before application

  • Signal should be reduced or eliminated if antibody is specific

• Multiple antibodies approach:

  • Use different antibodies targeting distinct FANCA epitopes

  • Consistent results across antibodies increase confidence in specificity

• Overexpression systems:

  • Compare signal in cells with and without FANCA overexpression

  • Tagged FANCA constructs can provide additional validation

• Patient-derived cell lines:

  • Cells from FA patients with FANCA mutations versus complemented versions

  • Example: "6914 pMMP-Empty versus 6914 pMMP-FANCA cells"

Ideally, combine multiple validation approaches to establish antibody specificity conclusively.

What are the proper storage and handling procedures for FANCA antibodies?

Proper storage and handling are essential for maintaining antibody performance:

• Storage conditions:

  • Most FANCA antibodies should be stored at -20°C

  • Avoid repeated freeze-thaw cycles by making small aliquots

  • Some formulations may contain 0.02% sodium azide and 50% glycerol

• Shipping and short-term storage:

  • Can be kept at 4°C for short periods (typically up to 1 month after reconstitution)

  • For longer storage (6+ months), maintain at -20°C to -70°C under sterile conditions

• Formulation considerations:

  • Some antibodies are supplied in PBS with preservatives

  • Small volume antibodies (20μL) may contain 0.1% BSA

• Reconstitution:

  • Follow manufacturer's specific instructions for reconstitution of lyophilized antibodies

  • Document reconstitution date on antibody vial

• Working dilutions:

  • Prepare fresh working dilutions on the day of experiment

  • Do not store diluted antibody for extended periods

Following these guidelines will help maintain antibody performance and reproducibility across experiments.

How can I optimize FANCA immunofluorescence protocols to visualize nuclear foci?

Visualizing FANCA nuclear foci requires careful optimization:

• Cell preparation:

  • Grow cells on poly-lysine coated slides

  • Induce DNA damage with appropriate agents (2mM hydroxyurea for 24 hours, mitomycin C at 100-200nM, or aphidicolin at 0.4-1.0μM)

• Pre-extraction and fixation:

  • Pre-extract with 0.5% TritonX-100 in PBS for 5 minutes at room temperature

  • Treat with hypotonic solution (PBS 0.4×) for 1 minute before fixation

  • Fix with 3.7-4% formaldehyde for 10 minutes

  • Additional permeabilization with 0.5% NP-40 in PBS after fixation

• Blocking and antibody incubation:

  • Block in 5% fetal bovine serum in PBS

  • Incubate with FANCA primary antibody (1:500 dilution) overnight at 4°C

  • Use appropriate fluorescent secondary antibodies (e.g., Alexa 488 anti-rabbit at 1:500)

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI

  • Mount with appropriate anti-fade mounting medium

  • For co-localization studies, include antibodies against other proteins (e.g., γ-H2AX at 1:3000)

• Imaging considerations:

  • Use confocal microscopy for better resolution of nuclear foci

  • Establish consistent criteria for quantifying foci (e.g., minimum size, intensity)

  • Include blinded scoring to avoid bias

This protocol has been successfully used to compare FANCA foci formation between normal and FA patient-derived cells, as well as to assess the impact of FANCA mutations on foci formation.

What methodologies are available for studying FANCA expression in clinical samples?

Several approaches can be used to assess FANCA expression in patient-derived samples:

• Western blot analysis:

  • Patient-derived lymphoblastoid cell lines or fibroblasts

  • Whole cell lysates prepared in appropriate lysis buffers

  • Gradient gels (3-8% Tris-Acetate) for better resolution

  • Detection with specific FANCA antibodies (e.g., Bethyl A301-980A at 1:500)

• ELISA:

  • For quantitative measurement of FANCA levels in serum

  • Commercial FANCA ELISA kits are available

  • Typical protocol includes:

    • Creating a standard curve (31.25-2000 pg/mL)

    • Diluting serum samples (1:2)

    • Incubating at 37°C for 1h 30min

    • Using biotin-labeled antibody and HRP-Streptavidin conjugate

• Immunohistochemistry:

  • For FANCA detection in tissue sections

  • Often requires antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0

  • Validated in various tissues including colon, cervical, and stomach cancer tissues

• Flow cytometry:

  • For detecting FANCA in blood cells or cell suspensions

  • Requires permeabilization for intracellular FANCA detection

• qPCR for FANCA mRNA expression:

  • As a complementary approach to protein detection

  • Useful for cases where antibody detection is challenging

The choice of method depends on the specific research question, sample availability, and required sensitivity.

How should I design experiments to evaluate FANCA mutants using antibodies?

For studying FANCA mutations and their functional consequences:

• Expression system selection:

  • Use FANCA-deficient cell lines complemented with wildtype or mutant FANCA

  • Common approaches include:

    • Retroviral vectors (e.g., pMMP)

    • Site-directed mutagenesis to create specific mutations

    • CRISPR-engineered cell lines

• Epitope considerations:

  • Ensure the antibody epitope is not affected by the mutation being studied

  • For example, antibodies targeting aa 1412-1431 won't detect mutations causing truncation before this region

  • Use antibodies targeting different regions when studying truncation mutations

• Functional assays:

  • Nuclear localization: Compare nuclear vs. cytoplasmic fractions by Western blot or IF

  • Foci formation: Quantify FANCA foci after DNA damage induction

  • FANCD2 monoubiquitination: Measure downstream pathway activation

  • Chromosomal breakage: Assess functional consequence of mutations

  • Protein-protein interactions: Determine if mutations disrupt complex formation

• Controls:

  • Wild-type FANCA as positive control

  • Empty vector as negative control

  • Known pathogenic and benign variants as references

• Experimental design:

  • Include treatments that induce DNA damage (e.g., MMC, APH, UV)

  • Use consistent cell densities and treatment times

  • Analyze multiple timepoints when relevant

This approach has been successfully used to characterize novel FANCA variants and determine their pathogenicity in Fanconi anemia research .

How are FANCA antibodies being used in cancer research?

FANCA antibodies have become increasingly important in cancer research:

• Biomarker studies:

  • FANCA expression levels as potential diagnostic or prognostic markers

  • ELISA-based detection of FANCA in patient serum samples

  • IHC analysis of FANCA in tumor tissues, including colon, cervical, and stomach cancers

• Head and neck squamous cell carcinoma (HNSCC) research:

  • Studying FANCA amplification in sporadic HNSCC

  • Comparing FANCA expression between FA-associated and sporadic tumors

  • Investigating FANCA's role in radioresistance mechanisms

• Lung adenocarcinoma studies:

  • FANCA as a potential immunotherapy target

  • Correlation between FANCA expression and immune infiltration levels

  • Prognostic value of FANCA expression in lung cancer

• Therapeutic development:

  • Identifying synthetic lethal interactions with FANCA deficiency

  • Screening compounds that selectively target FANCA-deficient cells

  • Developing FANCA-based immunotherapeutic approaches

• Cancer susceptibility research:

  • Studying the relationship between FANCA mutations and cancer predisposition

  • Investigating mechanisms by which FANCA deficiency promotes tumorigenesis

These applications demonstrate the value of FANCA antibodies in translational cancer research beyond their traditional use in Fanconi anemia studies.

What resources are available for Fanconi anemia researchers working with FANCA antibodies?

Several specialized resources support FA research:

• Fanconi Cancer Foundation (FCF) Repository:

  • Offers affinity-purified antisera against FANCA and other FA proteins

  • Part of the Fanconi Anemia Research Materials program in partnership with OHSU

  • Includes detailed antibody data sheets with validation information

• Cell line resources:

  • Immortalized human FA fibroblast cell lines for complementation groups including FANCA

  • HNSCC cell line trios: wild-type, FANCA knockout, and FANCA-complemented versions

  • FA cancer-derived cell pairs (FANC-mutant parent and complemented derivative)

• Research sharing requirements:

  • Users of FCF antibodies must submit use-data to support community research

  • Proper citation of the Fanconi Anemia Research Fund is required in publications

• Commercial antibody providers:

  • Multiple validated FANCA antibodies with different applications and epitopes

  • Technical support for optimization and troubleshooting

• Protocol repositories:

  • Detailed Western blot, IP, and IF protocols optimized for FANCA detection

  • Standardized functional assays for FANCA activity assessment

These resources facilitate reproducible and rigorous research in the Fanconi anemia field.

How can FANCA antibodies be used to study post-translational modifications?

FANCA undergoes several post-translational modifications that regulate its function:

• Phosphorylation analysis:

  • FANCA is activated by phosphorylation at specific sites (Ser849, 850, 858, and 1449)

  • Phospho-specific antibodies can be used to monitor FANCA activation status

  • Western blot analysis comparing total vs. phosphorylated FANCA before and after DNA damage

• Ubiquitination studies:

  • Immunoprecipitate FANCA using specific antibodies

  • Probe with anti-ubiquitin antibodies to detect ubiquitinated forms

  • Alternatively, use ubiquitin-specific affinity reagents followed by FANCA detection

• SUMOylation detection:

  • Similar to ubiquitination studies, but probing for SUMO modifications

  • May require denaturing conditions to preserve modifications

• Mass spectrometry approaches:

  • Immunoprecipitate FANCA from cells under different conditions

  • Subject to tryptic digestion and LC-MS/MS analysis

  • Identify and quantify modified peptides

• Cell fractionation experiments:

  • Separate nuclear and cytoplasmic fractions

  • Compare modification status of FANCA in different cellular compartments

  • Monitor changes in modification patterns after DNA damage induction

These approaches have revealed that post-translational modifications are critical for regulating FANCA localization, complex formation, and activity in the DNA damage response pathway.

What are the challenges in detecting endogenous versus overexpressed FANCA?

Researchers face different challenges when detecting native versus exogenous FANCA:

• Endogenous FANCA detection:

  • Lower expression levels may require more sensitive detection methods

  • Cell-type specific expression patterns affect detection thresholds

  • Higher antibody concentrations or enhanced chemiluminescence may be needed

  • Signal-to-noise ratio can be a challenge, requiring careful blocking optimization

  • Loading higher amounts of protein (30μg or more) for Western blot

• Overexpressed FANCA detection:

  • Expression levels may exceed physiological range

  • Potential artifacts from non-native expression patterns or interactions

  • May require lower antibody concentrations to avoid saturated signals

  • Tagged versions (HA-tagged FANCA) allow detection with anti-tag antibodies

  • Cellular localization may differ from endogenous protein

• Comparison considerations:

  • When comparing endogenous and overexpressed FANCA, load different amounts of lysate

  • Use gradient gels for better separation and resolution

  • Consider using fluorescent secondary antibodies for more quantitative comparisons

  • Include appropriate controls (e.g., FANCA-deficient cells, empty vector controls)

• Verification strategies:

  • Confirm identity of detected bands using knockout/knockdown controls

  • Validate using multiple antibodies targeting different FANCA epitopes

  • Correlate protein expression with mRNA levels when possible

These considerations are important for accurate interpretation of experimental results, particularly when studying the functional consequences of FANCA mutations or interactions.

How are FANCA antibodies being integrated with newer research technologies?

FANCA antibody applications are evolving with technological advances:

• Proximity proteomics:

  • BioID or APEX2 fusions to identify proteins in proximity to FANCA

  • Mass spectrometry identification of novel FANCA interactors

  • Requires validation with conventional co-IP using FANCA antibodies

• Live-cell imaging:

  • Combining immunofluorescence data with live-cell dynamics

  • Correlation of fixed-cell antibody staining with GFP-FANCA behavior

  • Super-resolution microscopy for detailed FANCA localization

• Single-cell analysis:

  • Flow cytometry with FANCA antibodies for heterogeneity assessment

  • Mass cytometry (CyTOF) incorporation of FANCA antibodies

  • Correlation with other cellular markers at single-cell resolution

• Spatial transcriptomics integration:

  • Combining FANCA protein detection with regional RNA expression

  • Understanding spatial context of FANCA expression in tissues

• Computational predictive modeling:

  • Using antibody-validated FANCA expression/localization data to inform models

  • Machine learning approaches to predict effects of FANCA variants

  • Similar to recent approaches used for antibody engineering

The integration of FANCA antibodies with these technologies is advancing our understanding of Fanconi anemia pathophysiology and FANCA's role in cancer development.

What is the significance of FANCA splice variants for antibody selection?

FANCA splice variants present important considerations for antibody selection:

• Known FANCA splice variants:

  • Several variants have been documented, including:

    • Truncation after Cys297

    • Deletion of aa 143-174 with truncation after Cys297

    • Alternative start site at Met528

    • Six aa substitution for aa 297-1455

  • Hypomorphic splice variant c.3624C>T (p.Ser1208=) impacts FA disease phenotype

• Epitope considerations:

  • Antibodies targeting different regions detect different splice variants

  • C-terminal antibodies (e.g., targeting aa 1400-C-terminus) won't detect truncated forms

  • N-terminal antibodies won't detect variants with alternative start sites

  • For comprehensive detection, use antibodies targeting multiple regions

• Experimental validation:

  • Western blot may reveal multiple bands corresponding to different isoforms

  • Observed band sizes may differ from predicted (163 kDa vs. observed 130, 75, 42 kDa)

  • Knockout/knockdown controls help confirm band identity

• Research implications:

  • Different variants may have distinct functional properties

  • Some variants may be tissue-specific or induced under specific conditions

  • Antibody selection should align with specific research questions

Understanding these considerations is crucial for accurately interpreting experimental results and avoiding false negatives when specific variants are relevant to the research question.

How can researchers contribute to improving FANCA antibody resources?

The research community can enhance FANCA antibody resources through:

• Data sharing:

  • Submit antibody use-data to repositories like the Fanconi Anemia Research Fund

  • Report detailed experimental conditions in publications

  • Document unexpected results or limitations observed with specific antibodies

• Validation standards:

  • Implement rigorous validation using genetic controls (knockout/knockdown)

  • Test antibodies across multiple applications and conditions

  • Perform epitope mapping to understand exact binding sites

• Protocol optimization:

  • Share optimized protocols for challenging applications

  • Document modifications that improve sensitivity or specificity

  • Provide troubleshooting guidance for common issues

• Cross-laboratory validation:

  • Participate in multi-laboratory testing of antibodies

  • Compare results across different experimental systems

  • Establish consensus on best practices

• Citation practices:

  • Properly acknowledge antibody sources in publications

  • Include catalog numbers and dilutions used

  • Follow recommended citation formats (e.g., "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.")

These collaborative efforts strengthen research reproducibility and advance the field of Fanconi anemia research.