FANCI Antibody

What is FANCI and why is it important in research?

FANCI is a 149.3 kDa protein consisting of 1328 amino acid residues that functions as a critical component in the Fanconi anemia (FA) pathway, which is essential for maintaining genomic stability. It plays a vital role in repairing DNA damage, particularly in response to double-strand breaks and DNA cross-links. FANCI is primarily localized in the nucleus, with some cytoplasmic presence, and exists in up to four different isoforms .

The protein is widely expressed across many tissue types and undergoes important post-translational modifications, including monoubiquitination and phosphorylation, which are crucial for its function in DNA repair . Research on FANCI is significant because defects in this protein can lead to Fanconi anemia, an autosomal recessive disorder characterized by bone marrow failure, congenital abnormalities, and increased susceptibility to cancer due to chromosomal instability .

What types of FANCI antibodies are available for research?

Several types of FANCI antibodies are available for research purposes:

• Monoclonal antibodies: Such as the mouse monoclonal IgG1 kappa light chain antibody (A-7) that detects FANCI protein of human origin .

• Polyclonal antibodies: Including rabbit polyclonal antibodies that target different epitopes of FANCI .

• Phospho-specific antibodies: Like the Anti-FANCI (Ser559) rabbit polyclonal antibody that specifically recognizes FANCI phosphorylated at Serine 559 .

• Conjugated antibodies: Available in various forms including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates for different detection methods .

These antibodies vary in their application suitability, with some optimized for Western blotting, others for immunoprecipitation, immunofluorescence, or ELISA .

How do I choose the right FANCI antibody for my experiment?

Selecting the appropriate FANCI antibody depends on several factors:

• Experimental application: Different antibodies are optimized for specific techniques. For Western blotting, antibodies like ab15344 and ab74332 have been validated and cited in publications . For immunoprecipitation, consider antibodies specifically tested for IP, such as 20789-1-AP or ab74332 .

• Species reactivity: Most commercially available FANCI antibodies are reactive to human samples, with some showing cross-reactivity with non-human primates . Verify the species reactivity matches your experimental model.

• Targeted epitope: Some research questions may require targeting specific regions or post-translational modifications of FANCI. For instance, if investigating the role of phosphorylation at Ser559, a phospho-specific antibody would be appropriate .

• Validation data: Review published literature citing the antibody and examine validation data provided by manufacturers, including Western blot images showing the expected ~149 kDa band .

• Clonality: Monoclonal antibodies offer high specificity for a single epitope, while polyclonal antibodies recognize multiple epitopes and may provide stronger signals but potentially more background .

What are the optimal conditions for Western blotting with FANCI antibodies?

For optimal Western blotting results with FANCI antibodies:

• Sample preparation: Nuclear extracts or whole cell lysates from human cell lines such as HeLa, HepG2, or Jurkat are recommended . Use freshly prepared samples when possible.

• Loading concentration: Load 5-50 μg of total protein depending on FANCI expression levels in your cell type .

• Dilution ranges:

  • For antibody ab15344: Use at 1/5000 dilution

  • For antibody ab74332: Use at 0.04 μg/mL

  • For antibody 20789-1-AP: Use at 1:1000-1:4000 dilution

  • For antibody from Abbexa: Use at 1/500-1/2000 dilution

• Electrophoresis conditions: Use SDS-PAGE with appropriate percentage gels (typically 6-8% for FANCI's large size of 149 kDa) .

• Transfer recommendations: Due to the large size of FANCI (149 kDa), longer transfer times or lower voltage/amperage may be needed to ensure complete transfer to membranes.

• Blocking: Typically use 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Detection system: ECL detection systems work well with appropriate exposure times (around 15 minutes has been reported for some antibodies) .

• Expected results: Look for a specific band at approximately 149 kDa .

How can I optimize immunofluorescence staining with FANCI antibodies?

For successful immunofluorescence staining with FANCI antibodies:

• Fixation: Use 4% paraformaldehyde for 15-20 minutes at room temperature, followed by permeabilization with 0.2% Triton X-100 for 10 minutes.

• Blocking: Block with 3-5% BSA or normal serum (matching the secondary antibody species) for 1 hour at room temperature.

• Primary antibody: Use FANCI antibody at the recommended dilution (e.g., FANCI Antibody A-7 has been validated for IF) . Incubate overnight at 4°C in a humidified chamber.

• Secondary antibody: Use species-appropriate fluorophore-conjugated secondary antibodies and incubate for 1-2 hours at room temperature in the dark.

• Counterstaining: DAPI can be used to visualize nuclei, which is particularly important since FANCI is predominantly nuclear.

• Expected pattern: FANCI typically shows nuclear localization with distinct foci formation particularly after DNA damage induction. After treatment with DNA crosslinking agents, FANCI forms nuclear foci that colocalize with other DNA repair proteins such as FANCD2 .

• Controls: Include negative controls (secondary antibody only) and positive controls (cell lines known to express FANCI).

What are the best approaches for immunoprecipitation of FANCI?

For effective immunoprecipitation of FANCI:

• Antibody selection: Use antibodies specifically validated for IP, such as Rabbit anti-FANCI Antibody from Bethyl Laboratories or antibody 20789-1-AP .

• Lysate preparation:

  • Use RIPA buffer or NP-40 lysis buffer supplemented with protease and phosphatase inhibitors.

  • For IP applications, use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate .

  • Cleared lysates should be pre-incubated with protein A/G beads to reduce non-specific binding.

• Immunoprecipitation protocol:

  • Incubate cleared lysate with FANCI antibody overnight at 4°C with gentle rotation.

  • Add protein A/G beads and incubate for 2-4 hours at 4°C.

  • Wash beads 4-5 times with IP wash buffer.

  • Elute the immunoprecipitated proteins with SDS sample buffer by heating at 95°C for 5 minutes.

• Co-immunoprecipitation considerations: FANCI has been shown to interact with PIDD1 specifically in cells treated with mitomycin C (MMC) and Chk1 inhibitor . For co-IP studies investigating FANCI interactions, consider appropriate treatment conditions to induce the interaction of interest.

• Controls: Always include IgG control to account for non-specific binding and input samples to evaluate IP efficiency.

How can FANCI antibodies be used to study the FANCI-FANCD2 complex formation?

The FANCI-FANCD2 (ID2) complex is central to the Fanconi anemia pathway. To study this complex:

• Co-immunoprecipitation: Use FANCI antibodies to pull down FANCI and detect co-precipitated FANCD2 by Western blotting. This approach can reveal the dynamics of complex formation under different conditions or treatments .

• Proximity ligation assay (PLA): This technique can detect protein-protein interactions in situ. Using FANCI and FANCD2 antibodies from different species, PLA can visualize and quantify the ID2 complex in fixed cells.

• Phosphorylation analysis: Phosphorylation of FANCI at Ser559 plays a key role in FANCI physically associating with FANCD2 and stabilizing the ID2 complex . Use phospho-specific antibodies (such as Anti-FANCI Ser559) to monitor this critical modification.

• Experimental considerations:

  • DNA damage induction: Treat cells with DNA crosslinking agents like mitomycin C to stimulate ID2 complex formation.

  • Time course analysis: The ID2 complex forms dynamically after DNA damage, so consider multiple time points.

  • Subcellular fractionation: Separate nuclear and chromatin-bound fractions to specifically examine the active complex at sites of DNA damage.

• Visualization: Immunofluorescence co-staining of FANCI and FANCD2 can reveal co-localization at DNA damage sites. This approach can be combined with other DNA damage markers (γH2AX, 53BP1) to confirm localization at damage sites.

What approaches can be used to study FANCI phosphorylation and its functional significance?

FANCI phosphorylation is critical for its function in DNA repair. To study this:

• Phospho-specific antibodies: Use antibodies that specifically recognize phosphorylated FANCI, such as Anti-FANCI (Ser559) . These antibodies allow detection of the phosphorylated form by Western blotting, immunofluorescence, or flow cytometry.

• Phosphatase treatment controls: To validate phospho-specific antibody specificity, treat samples with lambda phosphatase before analysis and compare signal with untreated samples.

• Kinase inhibitor studies:

  • ATM/ATR kinases phosphorylate FANCI at Ser556 and Ser559 .

  • Use specific inhibitors (ATM inhibitor KU-55933, ATR inhibitor VE-821) to determine the contribution of each kinase to FANCI phosphorylation.

• Phosphomimetic and phospho-dead mutants: Generate FANCI constructs with mutations at key phosphorylation sites:

  • Phosphomimetic (S559E/D) to mimic constitutive phosphorylation

  • Phospho-dead (S559A) to prevent phosphorylation

  These mutants can be expressed in FANCI-depleted cells to assess the functional consequences of phosphorylation.

• Functional assays: Measure DNA repair efficiency, cell survival after DNA damage, or FANCD2 monoubiquitination in cells expressing wild-type versus phospho-mutant FANCI to determine the functional significance of phosphorylation.

How can FANCI antibodies be employed to investigate the role of FANCI in the repair/apoptosis switch?

Recent research has revealed that FANCI functions as a repair/apoptosis switch in response to DNA damage . To investigate this dual role:

• FANCI-PIDD1 interaction studies:

  • Co-immunoprecipitation: Use FANCI antibodies to pull down endogenous FANCI and detect associated PIDD1, particularly after treatment with DNA crosslinking agents plus Chk1 inhibitor .

  • Immunofluorescence co-localization: Examine FANCI and PIDD1 co-localization using confocal microscopy. Research has shown that 43% of cells show colocalization between FANCI and phosphorylated PIDD1 (PIDD1pT788) after appropriate treatment .

• PIDDosome formation analysis:

  • Use FANCI antibodies in combination with PIDD1 and RAIDD antibodies to study the formation of the PIDDosome complex by size exclusion chromatography or co-IP approaches.

  • Compare PIDDosome assembly in wild-type versus FANCI-depleted cells to demonstrate its essential role in this process .

• Apoptosis assays in FANCI-manipulated systems:

  • Use FANCI antibodies to confirm knockdown or knockout efficiency in siRNA, shRNA, or CRISPR/Cas9-targeted cells.

  • Measure apoptotic markers (caspase activation, PARP cleavage, Annexin V staining) in these systems after DNA damage to determine how FANCI deficiency affects the apoptotic response.

• DNA binding-deficient FANCI mutants:

  • Research has shown that FANCI variants deprived of DNA binding (R1285Q or K294E/K339E) fail to interact with PIDD1 .

  • Use FANCI antibodies to examine the recruitment of these mutants to DNA damage sites and their impact on the repair/apoptosis decision.

What are common issues when using FANCI antibodies and how can they be resolved?

Researchers frequently encounter these issues when working with FANCI antibodies:

• No or weak signal in Western blot:

  • Ensure adequate protein loading (30-50 μg for whole cell lysates).

  • Verify transfer efficiency, especially for high molecular weight proteins like FANCI (149 kDa).

  • Consider alternative extraction methods; FANCI is nuclear and may require nuclear extraction protocols.

  • Optimize antibody concentration; try a range of dilutions around the recommended value.

  • Extend exposure time, as some FANCI antibodies have reported optimal exposure times of 15 minutes or more .

• Multiple bands or unexpected band size:

  • FANCI has multiple isoforms and undergoes post-translational modifications that can affect migration patterns.

  • Monoubiquitinated FANCI will appear at a higher molecular weight (~8-10 kDa shift).

  • Use positive controls (cell lines known to express FANCI) to verify band specificity.

  • Consider using FANCI knockout/knockdown samples as negative controls.

• High background in immunofluorescence:

  • Increase blocking time/concentration (try 5% BSA or normal serum).

  • Reduce primary antibody concentration.

  • Include additional washing steps with 0.1% Tween-20 in PBS.

  • Use highly cross-adsorbed secondary antibodies to reduce non-specific binding.

• Failed immunoprecipitation:

  • Increase antibody amount (try 2-4 μg for 1 mg lysate).

  • Optimize lysis buffer composition; add DNase I if DNA contamination is suspected.

  • Consider protein expression levels; FANCI expression may be cell-type dependent.

  • For co-IP of interacting partners, consider crosslinking to stabilize transient interactions.

How can I validate the specificity of my FANCI antibody?

Validating antibody specificity is crucial for reliable experimental results:

• Genetic approaches:

  • Use FANCI knockout/knockdown cells (siRNA, shRNA, or CRISPR/Cas9) as negative controls.

  • Compare with FANCI overexpression systems to confirm signal increase.

  • Patient-derived FA-I cells carrying FANCI mutations can serve as biological controls .

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide before application.

  • A specific antibody will show reduced or abolished signal when blocked with its cognate peptide.

• Multiple antibody validation:

  • Use different antibodies targeting distinct epitopes of FANCI.

  • Concordant results with multiple antibodies support specificity.

• Immunoprecipitation-mass spectrometry:

  • Perform IP with the FANCI antibody followed by mass spectrometry.

  • Specific antibodies should predominantly pull down FANCI and known interacting partners.

• Induction experiments:

  • DNA damage typically enhances FANCI recruitment to nuclear foci.

  • Treatment with mitomycin C should increase FANCI foci formation in immunofluorescence experiments.

How should I interpret changes in FANCI localization and post-translational modifications?

Proper interpretation of FANCI experimental data requires understanding its biology:

• Subcellular localization changes:

  • FANCI is predominantly nuclear but can shuttle between nucleus and cytoplasm.

  • After DNA damage, expect increased nuclear localization and formation of discrete nuclear foci.

  • Co-localization with FANCD2 at nuclear foci indicates active DNA repair complexes.

  • Failure to form nuclear foci after DNA damage may indicate defective DNA damage response or upstream signaling.

• Monoubiquitination:

  • FANCI monoubiquitination is detected as a band shift of ~8-10 kDa in Western blots.

  • This modification increases after DNA damage, particularly interstrand crosslinks.

  • Absence of monoubiquitination after DNA damage suggests defects in the FA pathway.

  • FANCD2 monoubiquitination often parallels FANCI monoubiquitination; compare both for comprehensive analysis.

• Phosphorylation:

  • Phosphorylation at Ser559 is critical for FANCI function in the FA pathway .

  • This modification increases after DNA damage and is mediated by ATM/ATR kinases.

  • Phosphorylation precedes and is required for efficient monoubiquitination.

  • Use phospho-specific antibodies to track this modification in response to different DNA damaging agents or in cells with mutations in upstream components.

• Protein-protein interactions:

  • FANCI interactions (with FANCD2, PIDD1, etc.) are often DNA damage-inducible.

  • The FANCI-PIDD1 interaction occurs specifically in response to interstrand crosslinks and not double-strand breaks .

  • FANCI's DNA binding ability is critical for some protein interactions; DNA binding-deficient mutants (R1285Q, K294E/K339E) fail to interact with PIDD1 .

What new roles of FANCI have been discovered beyond the classical FA pathway?

Recent research has uncovered novel functions of FANCI beyond its canonical role in DNA repair:

• Repair/Apoptosis switch function:

  • FANCI has been identified as a direct regulator of the repair/apoptosis decision in response to DNA damage.

  • It interacts with PIDD1 specifically after interstrand crosslink damage to promote PIDDosome formation and subsequent apoptosis when DNA repair is compromised .

  • FANCI enables PIDD1 phosphorylation by ATR and ATM, which is the initiating step in PIDDosome formation .

  • This represents a direct mechanism for how cells decide between repair and apoptosis pathways.

• Cell cycle checkpoint regulation:

  • FANCI participates in S phase and G2 phase checkpoint activation upon DNA damage .

  • This function may be independent of its role in the FA pathway and DNA repair.

• DNA binding properties:

  • FANCI specifically binds branched DNA structures, interacting with both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) .

  • This property may be important for recognition of specific DNA structures during replication stress or DNA damage.

• Emerging interactions with other cellular pathways:

  • Research has indicated connections between FANCI and ribosomal protein functions, suggesting potential roles beyond DNA repair .

  • A study has shown that inactivation of ribosomal protein S27-like impairs DNA interstrand cross-link repair by destabilization of FANCD2 and FANCI .

How can FANCI antibodies be used in cancer research and potential therapeutic development?

FANCI antibodies are valuable tools in cancer research:

• Biomarker studies:

  • FANCI expression and post-translational modifications can be assessed in tumor samples using immunohistochemistry with appropriate FANCI antibodies.

  • Altered FANCI expression has been observed in various cancers, and antibodies enable evaluation of its potential as a diagnostic or prognostic marker.

  • For immunohistochemistry applications, antibodies like 20789-1-AP have been validated for human intrahepatic cholangiocarcinoma tissue .

• Therapeutic response prediction:

  • FANCI antibodies can be used to monitor the FA pathway activation status in tumor samples.

  • This information may predict sensitivity to DNA crosslinking agents (cisplatin, mitomycin C) and PARP inhibitors, which show synthetic lethality with FA pathway defects.

• Drug development platforms:

  • High-content screening assays using fluorescently labeled FANCI antibodies can identify compounds that affect FANCI localization, post-translational modifications, or protein-protein interactions.

  • Such compounds might modulate DNA repair capacity and potentially sensitize cancer cells to existing therapies.

• Personalized medicine approaches:

  • Immunodetection of FANCI and its modifications in patient-derived xenografts or organoids can guide treatment selection.

  • FANCI antibodies enable monitoring of pathway activation during treatment, potentially identifying mechanisms of resistance.

• Synthetic lethality exploration:

  • FANCI antibodies can verify knockout/knockdown efficiency in synthetic lethality screens.

  • Recent research suggests connections between FANCI deficiency and colon cancer development via inhibition of DNA repair mechanisms , highlighting the importance of understanding FANCI's role in different cancer contexts.