FANCD2 Antibody

What is the molecular weight of FANCD2 protein and how does this affect antibody detection?

FANCD2 exists in two forms that can be detected by antibodies: the non-ubiquitinated form (S-form) at approximately 155-160 kDa and the monoubiquitinated form (L-form) at approximately 164-166 kDa . This size difference is crucial for experimental interpretation, as the ratio between these two forms often serves as a readout for Fanconi anemia pathway activation. When performing western blot analysis, using gradient gels (4-8%) with extended run times can help achieve better separation between these forms. Positive controls such as HeLa cells treated with DNA crosslinking agents like mitomycin C (MMC) are recommended to visualize both forms properly .

Which applications are most effectively supported by FANCD2 antibodies?

FANCD2 antibodies have been validated for multiple applications with varying success rates:

For most research applications, monoclonal antibodies targeting well-conserved epitopes show higher consistency in experimental results .

What species reactivity should I consider when selecting a FANCD2 antibody?

While most commercially available FANCD2 antibodies are raised against human FANCD2, many show cross-reactivity with mouse and rat orthologs due to sequence conservation . When working with non-human models, it's important to verify species reactivity:

• Human: All tested antibodies show reactivity

• Mouse: Select antibodies show reactivity, particularly those targeting N-terminal regions

• Rat: Limited reactivity, verification recommended

• Other species: Predicted based on sequence homology but requires validation

For cross-species studies, antibodies targeting the most conserved regions (particularly the N-terminal domain) offer better cross-reactivity .

How can I optimize detection of FANCD2 foci formation following DNA damage?

FANCD2 forms discrete nuclear foci at sites of DNA damage that can be visualized by immunofluorescence. Optimization strategies include:

• Damage induction: Treat cells with mitomycin C (MMC) at 100-500 ng/ml for 24h or ionizing radiation (IR) at 10 Gy followed by 6-8h recovery

• Fixation protocol: Use 4% paraformaldehyde for 10 minutes at room temperature (avoid methanol fixation which can disrupt foci morphology)

• Permeabilization: Critical step - use 0.3% Triton X-100 in PBS for 10 minutes

• Antibody selection: Use antibodies validated specifically for IF applications at 5 μg/ml

• Pre-extraction: Perform Triton pre-extraction (0.2% Triton X-100 in PBS for 2 minutes on ice) before fixation to remove soluble FANCD2 and enhance focus detection

• Counterstaining: Include γH2AX antibody to confirm colocalization with DNA damage sites

The visualization of FANCD2 foci is significantly improved when soluble nuclear proteins are removed before fixation, making pre-extraction a crucial step for quantitative analysis of foci formation .

What approaches are recommended for studying FANCD2-FANCI interactions?

The FANCD2-FANCI interaction forms the "ID complex" that is central to Fanconi anemia pathway function . Optimal approaches include:

• Co-immunoprecipitation:

  • Immunoprecipitate with either FANCD2 or FANCI antibodies

  • The interaction is robust with 15-20% of total FANCD2 recovered in FANCI immunoprecipitates

  • The interaction is DNA damage-independent

  • Both monoubiquitinated and non-ubiquitinated forms can be co-precipitated

• Proximity ligation assay:

  • More sensitive for detecting interactions in situ

  • Requires validated antibodies raised in different species

  • Allows visualization of interaction in different cellular compartments

• Immunofluorescence co-localization:

  • Both proteins form damage-induced foci that extensively colocalize

  • Use antibodies BL999 and BL1000 for FANCI and FI17 (sc-20022) for FANCD2

  • Include pre-extraction steps to improve visualization

• Bimolecular fluorescence complementation:

  • For studying dynamics of interaction in living cells

  • Requires expression of fusion constructs

Importantly, research has shown that the FANCD2-FANCI interaction occurs independently of FANCD2 monoubiquitination status, as both wild-type and K561R mutant FANCD2 co-precipitate with FANCI .

How do I troubleshoot inconsistent FANCD2 western blot results showing multiple bands?

Multiple bands in FANCD2 western blots can be caused by several factors:

For optimal FANCD2 detection in western blots:

• Use freshly prepared samples in RIPA buffer with both protease and phosphatase inhibitors

• Include 10 mM N-ethylmaleimide to prevent deubiquitination

• Use gradient gels (4-12%) with extended run times (>2 hours)

• Transfer to PVDF membranes (rather than nitrocellulose) for proteins of this size

• Include positive controls (HeLa cells treated with MMC) to confirm detection of both forms

How should I design experiments to study FANCD2 monoubiquitination as a readout for Fanconi anemia pathway activation?

Monoubiquitination of FANCD2 at lysine 561 is a key regulatory event in the Fanconi anemia pathway . To effectively study this modification:

• Experimental design considerations:

  • Include time course analysis (1-24h) following DNA damage induction

  • Test multiple DNA damaging agents (MMC, cisplatin, hydroxyurea, IR)

  • Include both positive controls (FA pathway-proficient cells) and negative controls (FA pathway-deficient cells like PD20)

  • Consider synchronized cell populations as monoubiquitination is cell cycle-regulated

• Detection methods:

  • Western blot: The most quantitative approach using antibodies that detect both S and L forms

  • Specific monoubiquitination-site antibodies: Some antibodies specifically recognize the ubiquitinated K561 site

  • Indirect measurement: Nuclear foci formation by IF correlates with monoubiquitination status

• Analysis considerations:

  • Quantify the L/S ratio rather than absolute levels

  • Normalize to loading controls

  • Consider cell cycle effects on basal monoubiquitination levels

  • Validate with genetic approaches (FANCD2 K561R mutant as negative control)

• Complementary approaches:

  • Co-IP with ubiquitin antibodies

  • Mass spectrometry for identification of ubiquitination site

  • CRISPR-Cas9 editing of K561 site as validation

The dual ubiquitin locking mechanism between FANCD2 and FANCI is particularly notable - ubiquitination of each protein is important for maintaining ubiquitination on the other, suggesting a complex interdependent regulation .

What controls are essential when validating FANCD2 antibody specificity for advanced applications?

Proper validation controls are critical for reliable FANCD2 antibody usage:

• Genetic controls:

  • FANCD2 knockout cell lines (strongest validation)

  • FANCD2 siRNA/shRNA knockdown cells (quantifiable reduction)

  • FANCD2-deficient patient cells (PD20 fibroblasts) vs. complemented lines

• Biochemical controls:

  • Peptide competition assays (particularly for polyclonal antibodies)

  • Immunoprecipitation followed by mass spectrometry verification

  • Detection of both monoubiquitinated and non-ubiquitinated forms

• Application-specific controls:

  • For IF: Secondary antibody-only controls; pre-absorption with immunizing peptide

  • For ChIP: IgG controls; input normalization; non-targeted genomic regions

  • For flow cytometry: Isotype controls; fluorescence-minus-one controls

• Cross-validation approaches:

  • Compare results from multiple antibodies recognizing different epitopes

  • Correlation between exogenous tagged constructs and endogenous protein detection

  • Confirmation of expected molecular weight shifts after post-translational modifications

For the most rigorous validation, comparing antibody reactivity in parental vs. FANCD2 knockout cell lines by western blot provides definitive confirmation of specificity, as demonstrated with several FANCD2 antibodies in the literature .

How can ChIP experiments be optimized to study FANCD2 recruitment to DNA damage sites?

FANCD2 chromatin immunoprecipitation (ChIP) requires specific optimization strategies:

• Sample preparation:

  • Crosslink with formaldehyde (1% for 10 minutes at room temperature)

  • Consider dual crosslinking (DSG followed by formaldehyde) for improved protein-DNA fixation

  • For site-specific DNA damage, use laser microirradiation or I-SceI-induced breaks

• Antibody selection:

  • Use ChIP-validated antibodies (reported in literature, PMID 28196964)

  • Monoclonal antibodies typically show less background than polyclonals

  • Epitope accessibility may be affected by crosslinking; test multiple antibodies

• Experimental conditions:

  • Sonication optimization is critical (aim for 200-500bp fragments)

  • Include FANCD2-deficient cells as negative controls

  • Consider sequential ChIP (FANCD2 followed by γH2AX) to identify damage-specific binding

• Data analysis:

  • Include input normalization and IgG controls

  • Compare enrichment at known FANCD2 binding sites versus random genomic regions

  • Validate key findings with alternative approaches (e.g., ChIP-re-ChIP)

• Advanced approaches:

  • ChIP-seq for genome-wide analysis of FANCD2 binding

  • CUT&RUN as an alternative to traditional ChIP with potentially lower background

  • Spike-in normalization for quantitative comparisons between conditions

The association of FANCD2 with chromatin is significantly enhanced after DNA damage, making the timing of sample collection post-damage a critical parameter for successful ChIP experiments .

How do I distinguish between FANCD2 and its paralog FANCI when they show similar molecular weights and functions?

FANCD2 and FANCI are paralogs that likely evolved from a common ancestral gene . Distinguishing between them requires careful experimental design:

• Antibody selection strategies:

  • Use antibodies raised against non-homologous regions (C-terminal domains show less conservation)

  • Validate antibody specificity using knockout/knockdown controls for each protein

  • Consider using epitope-tagged versions (HA-FANCI, FLAG-FANCD2) for clean discrimination

• Biochemical approaches:

  • FANCD2 migrates slightly faster than FANCI on SDS-PAGE (FANCD2: ~155-166 kDa; FANCI: slightly larger)

  • Both proteins undergo monoubiquitination, but at different lysine residues

  • Differential extraction protocols can help separate the proteins (FANCI may show nuclear rim staining in some experiments)

• Functional discrimination:

  • siRNA knockdown of each protein can reveal distinct phenotypes

  • FANCD2 ubiquitination is dependent on FANCI, but the reverse relationship is less strict

  • FANCD2 knockout affects FANCI foci formation more severely than vice versa

• Co-localization studies:

  • While both proteins co-localize extensively, super-resolution microscopy may reveal subtle differences in localization patterns

  • Timing of recruitment after damage may differ slightly

Understanding the reciprocal regulation between these proteins is important - FANCI depletion reduces FANCD2 monoubiquitination and steady-state levels, while FANCD2 depletion affects FANCI foci formation, highlighting their interdependent relationship .

What strategies should be employed when analyzing FANCD2 in primary patient samples with potential mutations?

Analyzing FANCD2 in patient samples presents unique challenges:

• Sample considerations:

  • Fresh samples yield better results than archival material

  • For blood samples, isolate mononuclear cells rather than whole blood

  • For tissue biopsies, consider laser capture microdissection for cell-type specificity

• Mutation-specific considerations:

  • Missense mutations may affect antibody epitope recognition

  • Truncating mutations may require N-terminal targeting antibodies

  • Consider RNA analysis (RT-PCR) alongside protein detection

• Methodological approaches:

  • Western blot: Most reliable for detecting both forms and potential abnormal sizes

  • Immunofluorescence: Assess foci formation capacity after DNA damage

  • Flow cytometry: For quantitative analysis in heterogeneous samples

• Control selection:

  • Age-matched healthy controls

  • Family members (particularly unaffected siblings)

  • Consider complementation with wild-type FANCD2 in patient-derived cells

• Functional readouts:

  • MMC sensitivity assays

  • Chromosomal breakage analysis

  • FANCD2 monoubiquitination status

  • Nuclear foci formation after DNA damage

When working with primary samples from potential Fanconi anemia patients, it's critical to assess both FANCD2 expression levels and functional activity (monoubiquitination and foci formation) as some mutations may allow protein expression but disrupt function .

How can phosphorylation states of FANCD2 be effectively studied in relation to ATR/ATM kinase activity?

FANCD2 is phosphorylated by ATR/ATM kinases in response to DNA damage, regulating its function . To study these modifications:

• Phosphorylation-specific approaches:

  • Phospho-specific antibodies: Limited commercial availability, may require custom development

  • Phos-tag SDS-PAGE: Allows separation of phosphorylated forms without specific antibodies

  • Lambda phosphatase treatment: To confirm phosphorylation-dependent mobility shifts

  • Mass spectrometry: For identification of specific phosphorylation sites

• Kinase manipulation strategies:

  • ATR/ATM inhibitors (VE-821, KU-55933)

  • Kinase-dead dominant negative constructs

  • siRNA-mediated knockdown of kinases

  • Phosphomimetic and phospho-dead mutants of key residues (e.g., S222)

• Functional correlation analyses:

  • Monitor monoubiquitination status along with phosphorylation

  • Assess chromatin binding and foci formation

  • Measure DNA repair capacity using reporter assays

  • Evaluate cell cycle checkpoint activation

• Advanced techniques:

  • Proximity ligation assays for detecting kinase-substrate interactions

  • FRET-based reporters for real-time kinase activity monitoring

  • Phosphoproteomics for global analysis

Research has established that ATR directly phosphorylates FANCD2 on several sites that are required for its function, including S222 . This phosphorylation appears to be a prerequisite for effective monoubiquitination and subsequent DNA repair activities, establishing a mechanistic link between DNA damage signaling and the Fanconi anemia pathway.

What emerging technologies are advancing FANCD2 research beyond traditional antibody applications?

Several cutting-edge approaches are revolutionizing FANCD2 research:

• CRISPR-based technologies:

  • Endogenous tagging of FANCD2 with fluorescent proteins or epitope tags

  • CUT&Tag for high-resolution chromatin localization

  • Base editing for studying specific post-translational modification sites

  • CRISPR screens for identifying novel FANCD2 regulators and interactors

• Live-cell imaging approaches:

  • FANCD2-FP fusions for real-time tracking of recruitment dynamics

  • FRAP (Fluorescence Recovery After Photobleaching) for mobility analysis

  • Single-molecule tracking for understanding FANCD2 behavior at individual damage sites

  • Optogenetic control of FANCD2 recruitment/function

• Structural biology advances:

  • Cryo-EM structures of the FANCD2-FANCI complex

  • Hydrogen-deuterium exchange mass spectrometry for conformational studies

  • Integrative structural modeling combining multiple data sources

• Single-cell technologies:

  • scRNA-seq combined with protein detection for correlating FANCD2 levels with transcriptional responses

  • Mass cytometry for comprehensive analysis of FANCD2 pathway activation in heterogeneous samples

  • Digital spatial profiling for tissue-level analysis of FANCD2 expression and activation