FAAP20 Antibody, FITC conjugated

What is FAAP20 and what cellular processes can be studied using FAAP20 antibodies?

FAAP20 is a 20 kDa protein (gene name: C1orf86 isoform 2; accession number: NP_872339.2) that functions as a unique component of the Fanconi anemia (FA) core complex involved in DNA repair processes . FAAP20 contains a conserved C-terminal ubiquitin-binding zinc finger (UBZ) domain that binds polyubiquitin chains and facilitates its recruitment to damaged chromatin via RNF8-mediated ubiquitination . The protein directly interacts with FANCA, another component of the FA core complex, and this interaction is critical for stabilizing FANCA in cells .

Using FAAP20 antibodies, researchers can study several key cellular processes:

For optimal results when using FITC-conjugated antibodies, researchers should consider that FAAP20's cellular abundance might be relatively low in some cell types, as noted in studies where FAAP20 protein was not readily detectable in HEK293t input samples without overexpression .

What are the recommended fixation and permeabilization protocols for FAAP20 immunofluorescence using FITC-conjugated antibodies?

When performing immunofluorescence studies with FITC-conjugated FAAP20 antibodies, proper fixation and permeabilization are critical for preserving both protein structure and fluorophore activity. Based on published FAAP20 localization studies, the following protocol is recommended:

• Fix cells with 4% paraformaldehyde in PBS for 15 minutes at room temperature

• Permeabilize with 0.3% Triton X-100 in PBS for 10 minutes at room temperature

• Block with 3% BSA in PBS containing 0.1% Triton X-100 for 1 hour

This protocol helps maintain FAAP20's nuclear localization pattern while providing adequate antibody accessibility. For visualizing FAAP20's recruitment to DNA damage sites, consider that studies have shown FAAP20 forms nuclear foci following DNA damage, particularly after treatment with DNA crosslinking agents like mitomycin C (MMC) .

When studying FAAP20's interaction with chromatin, it's important to note that FAAP20 recruitment to damaged chromatin occurs via RNF8-mediated ubiquitination . Therefore, treatments that disrupt this process might affect FAAP20 localization patterns observed with FITC-conjugated antibodies.

How can FAAP20 antibodies be used to detect changes in protein expression after DNA damage?

FAAP20 antibodies can effectively monitor changes in protein expression or localization following DNA damage, particularly after exposure to interstrand crosslinking agents. Western blotting and immunofluorescence represent complementary approaches for quantifying these changes:

Western Blotting Protocol:

• Treat cells with DNA-damaging agents (e.g., MMC at 100 ng/ml for 24 hours)

• Prepare whole cell lysates and fractionate into cytoplasmic and nuclear fractions

• Separate proteins by SDS-PAGE and perform standard Western blotting

• Detect FAAP20 using the appropriate antibody concentration

Immunofluorescence Protocol:

• Induce DNA damage (e.g., ionizing radiation or MMC treatment)

• Fix and permeabilize cells at different time points post-damage

• Stain with FITC-conjugated FAAP20 antibody

• Counterstain with DAPI and DNA damage markers (e.g., γH2AX)

Research has shown that FAAP20's interaction with FANCA occurs independently of mitomycin C (MMC) treatment , but its recruitment to damage sites increases following DNA damage. When quantifying immunofluorescence results, it's important to note that FAAP20 deficiency can lead to reduced FANCA protein levels , which might confound the interpretation of results if FANCA is used as a reference protein.

What controls should be included when using FAAP20 Antibody, FITC conjugated?

Proper controls are essential for accurate interpretation of results when using FITC-conjugated FAAP20 antibodies. The following controls should be included in experimental designs:

Specificity Controls:

• FAAP20 knockdown/knockout cells to verify antibody specificity

• Isotype-matched FITC-conjugated control antibody

• Blocking peptide competition (if available)

Technical Controls:

• Unstained samples to establish autofluorescence baseline

• Single-color controls for compensation in multicolor experiments

• Secondary antibody-only control when using indirect detection methods

Biological Controls:

• FANCA-deficient cells, as FAAP20 protein levels may be reduced in these cells

• Positive controls such as cells overexpressing FAAP20

• Time course after DNA damage to capture dynamic changes

Research has shown that FAAP20 knockdown results in decreased signal in pan-acetyl lysine immunoblots , suggesting that acetylation status may affect antibody recognition. Additionally, when performing co-localization studies, it's worth noting that FAAP20 forms a subcomplex with FANCA and potentially FANCG (the "AG20" complex) , which may influence its detection pattern.

How can FAAP20 Antibody, FITC conjugated be used to investigate FAAP20's role in homologous recombination independent of FANCA?

Recent research has revealed that FAAP20 has a marked role in homologous recombination (HR) at DNA double-strand breaks that is separable from its binding partner FANCA . FITC-conjugated FAAP20 antibodies can be strategically employed to investigate this independent function:

Experimental Design:

• Generate FANCA knockout cell lines using CRISPR-Cas9

• Confirm FANCA knockout by Western blotting and functional assays

• Induce DNA double-strand breaks using ionizing radiation or endonucleases

• Visualize FAAP20 recruitment using FITC-conjugated antibodies

• Quantify RAD51 foci formation as an HR readout

Research has shown that while FAAP20's role in homologous recombination is not dependent on FANCA, FAAP20 stimulates FANCA's biochemical activity in vitro and participates in the single-strand annealing pathway of double-strand break repair in a FANCA-dependent manner . This indicates that FAAP20 has roles in several homology-directed repair pathways.

When analyzing microscopy data, it's important to note that knockdown of each AG20 protein (FAAP20, FANCA, FANCG) causes a detectable decrease in chromatin-bound RAD51, with FAAP20 knockdown showing particularly pronounced effects .

What are the considerations for using FAAP20 Antibody, FITC conjugated in co-localization studies with other DNA repair proteins?

Co-localization studies using FITC-conjugated FAAP20 antibodies with other DNA repair proteins require careful experimental design:

Spectral Considerations:

• FITC emits in the green spectrum (peak ~520 nm), so choose complementary fluorophores for other targets

• Avoid spectrally overlapping dyes like GFP or Alexa Fluor 488

• Consider using far-red fluorophores for co-staining to minimize bleed-through

Biological Considerations:

• FAAP20 directly interacts with FANCA within the FA core complex

• FAAP20 has been shown to co-immunoprecipitate with RAD51

• FAAP20 is recruited to ICL sites and may co-localize with FANCD2

Methodology:

• Perform sequential staining if cross-reactivity is a concern

• Use appropriate blocking to reduce non-specific binding

• Include single-stained controls for accurate compensation

• Analyze co-localization using quantitative metrics (Pearson's correlation, Manders' coefficients)

Research has demonstrated that FAAP20 knockdown reduces nuclear RAD51 Irradiation-induced foci and sensitizes cancer cells to ionizing radiation and PARP inhibition . When designing co-localization experiments, it's worth noting that FAAP20 binds to the middle region of FANCA (specifically regions corresponding to deletion mutants FANCA-D3 and FANCA-D4) , which may affect epitope accessibility in certain experimental conditions.

How can FAAP20 Antibody, FITC conjugated be used to study post-translational modifications of FAAP20?

FAAP20 undergoes several post-translational modifications that regulate its function, including acetylation and ubiquitination. FITC-conjugated FAAP20 antibodies can be used to study these modifications:

Acetylation Studies:
Research has shown that FAAP20 can be acetylated, mediated by p300/CBP acetyltransferases . To study this modification:

• Treat cells with histone deacetylase inhibitors (e.g., trichostatin A)

• Perform immunoprecipitation with FAAP20 antibody

• Probe for acetylation using pan-acetyl lysine antibodies

• Alternatively, use modification-specific antibodies if available

Ubiquitination Studies:
FAAP20 contains a ubiquitin-binding zinc finger (UBZ) domain and undergoes regulated proteolysis:

• Treat cells with proteasome inhibitors (e.g., MG132)

• Immunoprecipitate FAAP20 using appropriate antibodies

• Probe for ubiquitination using anti-ubiquitin antibodies

When studying these modifications, it's important to note that knockdown of p300 and CBP has been shown to affect FAAP20 acetylation . Additionally, although FAAP20 contains a ubiquitin-binding domain, studies did not observe appreciable binding of FAAP20 with monoubiquitinated FANCD2 in vitro , suggesting specificity in its interactions with ubiquitinated proteins.

What methodologies can be used to investigate FAAP20's functional relationship with the FA core complex using FITC-conjugated antibodies?

FAAP20's relationship with the FA core complex can be investigated using several methodologies with FITC-conjugated antibodies:

Chromatin Immunoprecipitation (ChIP) Analysis:

• Cross-link proteins to DNA using formaldehyde

• Immunoprecipitate with FITC-conjugated FAAP20 antibody

• Analyze enrichment at ICL sites or double-strand breaks

• Compare with ChIP using antibodies against other FA proteins

Research has demonstrated that FAAP20 shows approximately fourfold enrichment at cross-linked DNA substrates using eChIP assays with defined ICLs , suggesting its direct involvement at damage sites.

Proximity Ligation Assay (PLA):
This technique can visualize protein-protein interactions in situ:

• Use FITC-conjugated FAAP20 antibody with antibodies against other FA proteins

• Follow PLA protocol to detect interactions within 40 nm proximity

• Quantify PLA signals before and after DNA damage induction

Sequential Chromatin Immunoprecipitation (Re-ChIP):
To identify co-occupancy of FAAP20 with other FA proteins:

• Perform first ChIP with FAAP20 antibody

• Elute complexes and perform second ChIP with antibodies against other FA core complex proteins

• Analyze enrichment patterns to determine co-occupancy

When interpreting results, consider that FAAP20-deficient cells display defects in FANCD2 monoubiquitination and FANCD2 foci formation following MMC treatment . Additionally, FAAP20 loss causes a reduction in FANCA protein levels , which may complicate the interpretation of results when studying other FA core complex components.

How can live-cell imaging be performed using FAAP20 antibody fragments conjugated with FITC?

Live-cell imaging presents unique challenges when studying intracellular proteins like FAAP20. Using FITC-conjugated antibody fragments offers one approach:

Methodology:

• Generate Fab fragments from FAAP20 antibodies

• Conjugate with FITC using standard protocols

• Introduce into living cells using microinjection or cell-penetrating peptides

• Perform time-lapse microscopy before and after DNA damage induction

Technical Considerations:

• Antibody fragments must retain specificity and not disrupt FAAP20 function

• Cell delivery methods should minimize cellular stress

• Photobleaching of FITC must be minimized using anti-fade reagents

• Signal-to-noise ratio may be challenging given FAAP20's potentially low abundance

Alternative Approaches:

• CRISPR-Cas9 knock-in of fluorescent tags (e.g., GFP) to endogenous FAAP20

• Expression of fluorescently-tagged FAAP20 with simultaneous knockdown of endogenous protein

When designing live-cell experiments, consider that FAAP20 interacts with FANCA, which occurs independently of mitomycin C (MMC) treatment , suggesting a constitutive interaction that might be observable even in unstressed conditions. Additionally, FAAP20's recruitment to damaged chromatin via RNF8-mediated ubiquitination suggests that observing its dynamics following DNA damage may provide valuable insights into its function.

What methodological approaches can distinguish FAAP20's roles in different DNA repair pathways?

FAAP20 participates in multiple DNA repair pathways, including ICL repair and homologous recombination. FITC-conjugated antibodies can help distinguish its roles:

Pathway-Specific Induction:

• Induce ICLs using agents like mitomycin C or cisplatin

• Create double-strand breaks using ionizing radiation or endonucleases

• Track FAAP20 recruitment using FITC-conjugated antibodies

• Compare timing, intensity, and co-localization patterns

Genetic Background Manipulation:

• Generate knockouts of pathway-specific factors (e.g., BRCA2 for HR, FANCA for ICL repair)

• Assess FAAP20 recruitment and function in these backgrounds

• Complement with wild-type or mutant proteins to restore function

Research has shown that FAAP20 has roles in homologous recombination that are independent of FANCA, while its participation in single-strand annealing is FANCA-dependent . The table below summarizes key experimental approaches:

When interpreting results, it's important to note that although FAAP20's role in homologous recombination is not dependent on FANCA, FAAP20 stimulates FANCA's biochemical activity in vitro , suggesting complex interplay between these proteins across different repair contexts.