FAAP20 Antibody, HRP conjugated

What is FAAP20 and why is it important in DNA repair research?

FAAP20 (Fanconi Anemia Associated Protein 20) is a critical component of the Fanconi anemia (FA) pathway that associates with the FA core complex. It plays essential roles in promoting FANCD2/FANCI monoubiquitination and activating the damage response to interstrand crosslink (ICL) damage. Beyond its established role in the FA pathway, FAAP20 has recently been identified as having significant functions in homologous recombination (HR) at DNA double-strand breaks (DSBs) not associated with ICLs . This dual functionality makes FAAP20 an important research target for understanding both the FA pathway and broader DNA repair mechanisms.

Research has demonstrated that FAAP20 has surprisingly extensive roles in homology-directed repair (HDR) pathways of DSB repair that operate through both FANCA-dependent and FANCA-independent mechanisms . Loss of FAAP20 inhibits cell growth in response to DSB damage and PARP inhibition, highlighting its potential significance in cancer biology and therapeutic resistance .

What are the optimal immunoblotting conditions for FAAP20 antibody?

Based on established protocols in the literature, optimal immunoblotting conditions for FAAP20 antibody include:

• Blocking: Use 5% milk in PBS + 0.2% Tween-20 for 1 hour at room temperature

• Primary antibody incubation: Dilute FAAP20 antibody 1:2000 in blocking solution and incubate overnight at 4°C

• Washing: Perform multiple washes with PBS + 0.2% Tween-20

• Secondary antibody: For HRP-conjugated secondary antibodies, use a 1:5000 dilution and incubate for 1 hour at room temperature

• Detection: Use ECL detection reagent for visualization

• Analysis: Band intensity can be quantified using ImageJ software

When optimizing your protocol, consider that FAAP20 protein may have low abundance in some cell lines. In HEK293t cells, for example, researchers have noted that endogenous FAAP20 was not detectable in input samples due to low protein abundance and excessive dilution .

How can I effectively use FAAP20 antibody in immunofluorescence microscopy?

For effective immunofluorescence microscopy using FAAP20 antibody:

• Fixation: Fix cells in methanol at -20°C for 30 minutes or use 4% paraformaldehyde

• Permeabilization: If using paraformaldehyde, permeabilize with 0.5% Triton X-100

• Blocking: Block in PBS containing 3% BSA

• Primary antibody: Apply FAAP20 antibody at appropriate dilution (typically 1:2000 based on published protocols) and incubate overnight at 4°C

• Secondary antibody: Use fluorescent-conjugated secondary antibodies (e.g., Rhodamine-conjugated Donkey anti-Rabbit IgG at 1:500 dilution)

• Co-staining: For co-localization studies, FAAP20 can be effectively co-stained with other DNA repair proteins such as FANCD2, PALB2, or γH2AX

• Visualization: Analyze using appropriate fluorescence microscopy

When designing co-localization experiments, consider that FAAP20 and FANCD2 show strong co-localization in response to MMC treatment, whereas FAAP20 and other repair proteins may exhibit different patterns depending on the type of DNA damage .

What are the recommended controls when studying FAAP20 in DNA repair pathways?

When studying FAAP20 in DNA repair pathways, include these critical controls:

• Positive controls:

  • Include cell lines with known expression of FAAP20 (e.g., HeLa cells)

  • Use DNA damaging agents known to activate the FA pathway (MMC, cisplatin)

  • Include analysis of known interacting partners (FANCA, FANCD2)

• Negative controls:

  • FAAP20-depleted cells via siRNA or shRNA (validate using multiple target sequences)

  • Isotype control antibodies for immunoprecipitation experiments

  • Cell cycle markers to distinguish cell cycle-dependent effects

• Validation controls:

  • Use alternative siRNA/shRNA sequences targeting different regions of FAAP20 mRNA

  • For example, researchers have confirmed results using shRNA targeting a different sequence, resulting in reduced FAAP20 levels and a significant decrease in FANCD2 foci

  • 53BP1 has been used as a negative control in RAD51 immunoprecipitation experiments when probing for FAAP20 interaction

• Functional controls:

  • Assess both FANCD2 and PALB2 foci formation as readouts for different aspects of the FA pathway

  • Include γH2AX as a marker for DSBs

How should I design RNAi experiments to study FAAP20 function?

For effective RNAi experiments targeting FAAP20:

• Design multiple target sequences: Use at least two independent siRNA or shRNA sequences targeting different regions of FAAP20 mRNA to confirm specificity of phenotypes.

• Validate knockdown efficiency:

  • Confirm FAAP20 depletion by immunoblotting (1:2000 antibody dilution)

  • Assess protein levels at 48-72 hours post-transfection

  • Include appropriate loading controls (e.g., actin at 1:5000 dilution)

• Functional validation:

  • Assess FANCD2 foci formation in MMC-treated cells (FAAP20 depletion reduces FANCD2 foci)

  • Evaluate RAD51 foci formation (FAAP20 knockdown reduces nuclear RAD51 foci)

  • Measure HR efficiency using reporter assays

• Controls and complementation:

  • Include non-targeting siRNA/shRNA controls

  • Consider rescue experiments with siRNA-resistant FAAP20 constructs

  • Compare with knockdown of other FA proteins (FANCA, FANCD2) to distinguish specific functions

Published studies have demonstrated that FAAP20 depletion results in a marked reduction in FANCD2 foci following MMC treatment, while not significantly affecting PALB2 foci . This selective effect highlights the importance of examining multiple downstream factors.

What are the recommended antibody dilutions for different experimental applications?

Based on published protocols, the following dilutions are recommended for FAAP20 antibody applications:

For other antibodies commonly used in FAAP20-related studies:

• FANCD2: 1:2000 for immunoblotting

• RAP80: 1:4000 for immunoblotting

• Actin: 1:5000 for immunoblotting (loading control)

• RNF8: 1:400 for immunoblotting

• MDC1: 1:1000 for immunoblotting

How does FAAP20 contribute to homologous recombination independently of FANCA?

FAAP20 has been found to play a substantial role in homologous recombination (HR) that is separable from its binding partner FANCA. Key findings include:

• Independent HR function: FAAP20 knockdown causes a more profound decrease in HR compared to FANCA knockdown, suggesting non-redundant functions in the HR pathway .

• Impact on RAD51: FAAP20 loss causes a reduction in nuclear RAD51 irradiation-induced foci, similar to other known HR factors. Western blotting of chromatin fractions shows that FAAP20 knockdown causes a detectable decrease in chromatin-bound RAD51, though global RAD51 protein levels remain unaffected .

• Direct interaction with RAD51: Co-immunoprecipitation experiments have demonstrated that FAAP20 is present in RAD51-immunoprecipitated fractions but not in IgG control fractions, suggesting a physical interaction between FAAP20 and RAD51 .

• Comparison with FANCD2: While FANCD2 depletion does cause a substantial decrease in HR events (~75-80% decrease), HR events are still significantly higher in FANCD2-knockdown cells compared to FAAP20-knockdown cells, indicating FAAP20 may have a more prominent role than its FA partners in certain HR contexts .

This independent function in HR may explain why FAAP20 loss, like other known HR factors, inhibits cell growth in response to DSB damage or PARP inhibition—a phenotype likely not attributable to its role in single-strand annealing (SSA) .

What is the relationship between FAAP20 and FANCA in DNA repair pathways?

The relationship between FAAP20 and FANCA in DNA repair pathways is complex, with both cooperative and independent functions:

• Biochemical stimulation: FAAP20 dramatically stimulates FANCA's strand annealing and exchange activities by increasing FANCA's binding affinity to nucleic acid substrates. This stimulation is similar to FANCG's ability to enhance FANCA's biochemical activities .

• SSA pathway dependence: FAAP20 participates in the single-strand annealing (SSA) pathway of double-strand break repair in a FANCA-dependent manner, indicating collaboration between these proteins in certain repair contexts .

• Non-redundant HR functions: Despite their interaction, FAAP20 and FANCA display non-redundant functions in HR repair. FAAP20 knockdown causes a more profound decrease in HR compared to FANCA knockdown .

• Mutual stabilization: FAAP20 and FANCG stabilize FANCA protein in cells, while FANCA reciprocally stabilizes FAAP20 and FANCG .

• AG20 subcomplex: While FAAP20 is proposed to form the "AG20 subcomplex" with FANCA and FANCG, experimental evidence suggests that simultaneous binding of FANCA to both FANCG and FAAP20 is not required for its DNA processing roles. FAAP20's repair roles with FANCA appear separable from FANCA's repair roles with FANCG .

This complex relationship suggests that FAAP20 has both FANCA-dependent and FANCA-independent functions in different DNA repair pathways, with its roles in SSA likely involving FANCA whereas its roles in HR are not redundant with FANCA .

How does FAAP20 depletion affect cellular sensitivity to DNA damaging agents?

FAAP20 depletion affects cellular sensitivity to DNA damaging agents in several ways:

• Radiation sensitivity: FAAP20 loss sensitizes cancer cells to ionizing radiation, consistent with its role in homologous recombination repair of DNA double-strand breaks .

• PARP inhibitor sensitivity: Cells with FAAP20 depletion show increased sensitivity to PARP inhibition, similar to the effects seen with deficiencies in other homology-directed repair factors .

• Interstrand crosslink response: FAAP20 depletion results in a marked reduction in the percentage of cells with FANCD2 foci following treatment with mitomycin C (MMC), an interstrand crosslinking agent. This indicates impaired activation of the FA pathway in response to ICL damage .

• Growth inhibition: Like other known HR factors, FAAP20 loss inhibits cell growth in response to DSB damage or PARP inhibition, highlighting its importance in cellular proliferation following DNA damage .

• Comparison with other FA proteins: While FAAP20 depletion affects FANCD2 foci formation after MMC treatment, it does not significantly affect PALB2 foci. This contrasts with RAP80 depletion, which reduces PALB2 foci but not FANCD2 foci, indicating specificity in the roles of different ubiquitin-binding proteins in the DNA damage response .

These findings have potential implications for cancer therapy, as FAAP20 status may influence tumor response to radiation therapy and PARP inhibitors, similar to other DNA repair factors like BRCA1/2.

What methods can be used to study FAAP20's interactions with other repair proteins?

Several methods have been successfully employed to study FAAP20's interactions with other repair proteins:

• Co-immunoprecipitation (Co-IP):

  • Has been used to demonstrate interaction between FAAP20 and RAD51

  • Pull-down of RAD51 using specific anti-RAD51 antibody in HEK293t cells with overexpressed FAAP20 showed FAAP20 present in immunoprecipitated fractions

  • Important to include isotype control (e.g., rabbit IgG) and negative control proteins (e.g., 53BP1)

• Immunofluorescence co-localization:

  • Demonstrates spatial proximity of FAAP20 with other repair proteins at sites of DNA damage

  • FAAP20 shows strong co-localization with FANCD2 after MMC treatment

  • Co-localization analysis with γH2AX can determine association with DSBs

• Chromatin fractionation:

  • Western blotting of chromatin fractions can reveal recruitment of FAAP20 and other repair proteins to chromatin

  • Has shown that FAAP20 knockdown causes decreased RAD51 recruitment to chromatin

• RNAi-based epistasis analysis:

  • Depletion of FAAP20 and other repair proteins individually or in combination can reveal functional relationships

  • FAAP20 depletion reduces FANCD2 foci but not PALB2 foci after MMC treatment

  • RAP80 depletion reduces PALB2 foci but not FANCD2 foci

• In vitro biochemical assays:

  • Electrophoretic mobility shift assays (EMSA) with purified proteins to assess DNA binding

  • Strand exchange and annealing assays to evaluate effect of FAAP20 on FANCA's biochemical activities

  • These assays have shown that FAAP20 stimulates FANCA's DNA binding, strand exchange, and strand annealing activities

How can I improve detection of low-abundance FAAP20 in Western blots?

For improving detection of low-abundance FAAP20 in Western blots:

• Sample preparation optimization:

  • Use cell lysis buffers containing protease inhibitors to prevent degradation

  • Consider enrichment techniques such as immunoprecipitation or chromatin fractionation

  • Note that in HEK293t cells, endogenous FAAP20 was not detectable in input samples due to low protein abundance

• Protein loading and transfer:

  • Increase total protein loaded per lane (50-100 μg may be necessary)

  • Optimize transfer conditions for proteins in FAAP20's molecular weight range

  • Consider using PVDF membranes which may offer better protein retention than nitrocellulose

• Antibody optimization:

  • Increase primary antibody concentration (try 1:1000 instead of 1:2000)

  • Extend primary antibody incubation time (overnight at 4°C is standard)

  • Consider using signal enhancement systems compatible with HRP-conjugated antibodies

• Detection enhancement:

  • Use high-sensitivity ECL substrates designed for detecting low-abundance proteins

  • Extend exposure times when imaging

  • Consider using cooled CCD camera systems for digital imaging rather than film

• Positive controls:

  • Include lysates from cells overexpressing FAAP20 as a positive control

  • Alternatively, use cell lines known to express higher levels of FAAP20

What are the best approaches for analyzing FAAP20 foci formation after DNA damage?

For optimal analysis of FAAP20 foci formation after DNA damage:

• DNA damage induction:

  • Mitomycin C (MMC) treatment effectively induces FAAP20 and FANCD2 foci

  • Ionizing radiation can be used to study FAAP20's role in DSB repair

  • Consider time-course experiments to capture optimal foci formation (typically 6-24 hours post-damage)

• Immunofluorescence protocol optimization:

  • Methanol fixation at -20°C for 30 minutes has been successfully used

  • Use primary antibody at 1:2000 dilution with overnight incubation at 4°C

  • Include co-staining with γH2AX as a marker for DSBs

• Quantification methods:

  • Count cells with ≥5 or ≥10 foci per nucleus (threshold should be established based on background in untreated cells)

  • Score at least 200 cells per condition to ensure statistical significance

  • Use ImageJ or CellProfiler for automated foci quantification

• Controls and comparisons:

  • Include both untreated and treated controls

  • Compare FAAP20 foci with FANCD2 and PALB2 foci formation

  • Analysis of FAAP20-deficient cells and cells with other FA protein deficiencies provides valuable comparisons

• Advanced techniques:

  • Laser microirradiation can be used to study real-time recruitment of FAAP20 to sites of DNA damage

  • Live-cell imaging with fluorescently tagged FAAP20 can reveal dynamics of recruitment and resolution of foci

How can I optimize co-immunoprecipitation protocols for studying FAAP20 interactions?

To optimize co-immunoprecipitation protocols for studying FAAP20 interactions:

• Cell lysis optimization:

  • Use buffers containing 0.5% NP-40 or 0.1% Triton X-100 to maintain protein-protein interactions

  • Include protease inhibitors, phosphatase inhibitors, and potentially deubiquitinase inhibitors

  • Consider benzonase treatment to reduce DNA-mediated interactions

• Antibody selection and validation:

  • Test multiple antibodies for their efficiency in immunoprecipitation

  • Consider epitope tags (HA, FLAG, etc.) for overexpression studies if antibody quality is an issue

  • Validate antibody specificity using FAAP20-depleted cells as negative controls

• Immunoprecipitation conditions:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Optimize antibody amount (typically 2-5 μg per mg of total protein)

  • Extend incubation time (overnight at 4°C) to enhance weak interactions

• Enhancing detection of transient interactions:

  • Consider crosslinking approaches (formaldehyde, DSS) for capturing transient interactions

  • DNA damage treatment may enhance certain interactions

  • For DNA-dependent interactions, modify nuclease treatment accordingly

• Controls and verification:

  • Always include isotype control antibodies (e.g., rabbit IgG for rabbit FAAP20 antibodies)

  • Include negative control proteins (e.g., 53BP1 has been used as a negative control in RAD51 IPs)

  • For overexpression studies, empty vector transfection serves as a control

• Special considerations for FAAP20:

  • Due to low abundance, overexpression may be necessary for detection in IP experiments

  • When studying interactions with FANCA, consider that FAAP20 and FANCG may compete for binding

What are the emerging roles of FAAP20 beyond the canonical FA pathway?

Recent research has revealed several emerging roles for FAAP20 beyond its canonical function in the Fanconi anemia pathway:

• Independent homologous recombination function: FAAP20 has a marked role in homologous recombination at DNA double-strand breaks not associated with interstrand crosslinks, which is separable from its binding partner FANCA .

• Multiple homology-directed repair pathways: FAAP20 participates in several homology-directed repair pathways, including both HR and single-strand annealing (SSA) .

• RAD51 regulation: FAAP20 loss causes a reduction in nuclear RAD51 irradiation-induced foci, and co-immunoprecipitation experiments suggest a physical interaction between FAAP20 and RAD51, indicating a potential role in RAD51 regulation .

• Cell growth and proliferation: FAAP20 supports cellular colony formation and proliferation, corresponding with its strong HR function. This may underlie FAAP20's potential to affect cancer outcomes and therapy resistance .

• Therapeutic relevance: FAAP20 loss sensitizes cancer cells to ionizing radiation and PARP inhibition, suggesting potential therapeutic implications in cancer treatment strategies .

• Ubiquitin signaling network: FAAP20 functions in an ubiquitin signaling network that includes the RNF8 E3 ligase and distinct ubiquitin-binding proteins, coordinating the recruitment of various DNA repair proteins .

These emerging functions suggest that FAAP20 may have broader significance in genome maintenance and cancer biology than previously appreciated, making it an important target for continued investigation.

How might advanced techniques improve our understanding of FAAP20 functions?

Advanced techniques that could enhance our understanding of FAAP20 functions include:

• CRISPR-Cas9 genome editing:

  • Generation of FAAP20 knockout cell lines for cleaner functional studies compared to RNAi

  • Creation of endogenously tagged FAAP20 (e.g., GFP-FAAP20) for live-cell imaging without overexpression artifacts

  • Introduction of specific mutations to map functional domains and post-translational modification sites

• Proximity labeling proteomics:

  • BioID or APEX2 fusion to FAAP20 to identify proximal proteins in living cells

  • TurboID-based approaches for rapid labeling to capture dynamic interactions after DNA damage

  • Compare interactomes in different damage conditions and cell cycle phases

• Single-molecule imaging:

  • Super-resolution microscopy to visualize FAAP20 localization at DNA damage sites with nanometer precision

  • Single-particle tracking to monitor FAAP20 dynamics at sites of DNA damage

  • FRET-based approaches to study protein-protein interactions in live cells

• Structural biology approaches:

  • Cryo-EM analysis of FAAP20 within the FA core complex

  • Structural studies of FAAP20's interactions with ubiquitin and other binding partners

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

• Genomic mapping techniques:

  • ChIP-seq to map FAAP20 binding sites across the genome

  • BLESS or END-seq to correlate FAAP20 binding with sites of DNA damage

  • Integration with other genomic datasets to understand context-specific functions

• Patient-derived models:

  • Analysis of FAAP20 functions in patient-derived cells with FA pathway deficiencies

  • Correlation of FAAP20 expression or mutations with clinical outcomes in cancer patients

  • Development of patient-derived organoids to study FAAP20 in a more physiological context

These advanced approaches could provide deeper insights into FAAP20's molecular functions, its regulation in response to different types of DNA damage, and its potential as a therapeutic target.