Phospho-FANCA (S1149) Antibody

What is Phospho-FANCA (Ser1149) Antibody and what specific epitope does it recognize?

Phospho-FANCA (Ser1149) Antibody is a rabbit polyclonal antibody specifically designed to detect the FANCA protein only when phosphorylated at serine residue 1149. This antibody has been generated using synthetic phosphopeptides derived from human FANCA around the phosphorylation site of Serine 1149 as immunogens . The antibody undergoes affinity purification from rabbit antiserum by affinity-chromatography using epitope-specific immunogen, achieving a purity greater than 95% as determined by SDS-PAGE . This high specificity allows researchers to distinguish between phosphorylated and non-phosphorylated forms of FANCA, which is essential for studying signaling pathways and post-translational modifications affecting this protein's function.

What applications is Phospho-FANCA (Ser1149) Antibody validated for?

Phospho-FANCA (Ser1149) Antibody has been validated for multiple research applications, providing versatility in experimental design. The antibody is confirmed suitable for Western blotting (WB), immunohistochemistry (IHC), immunocytochemistry/immunofluorescence (ICC/IF), and immunoprecipitation (IP) . For Western blot applications, the antibody has demonstrated successful detection using K562 whole cell lysates . While these applications have been validated, researchers should note that optimal working dilutions should be determined experimentally for each specific experimental setup. Suggested starting dilutions include 1:500-1:2000 for Western blotting, 1:100-1:300 for immunohistochemistry on paraffin-embedded sections, and 1:200-1:1000 for immunofluorescence applications .

What is the molecular weight of the FANCA protein detected by this antibody?

The molecular weight of FANCA protein detected by this antibody is approximately 162-163 kDa. This is consistent across multiple product datasheets, with both calculated and observed molecular weights falling within this range . When performing Western blot analysis, researchers should expect to observe a band at approximately 163 kDa, which represents the phosphorylated form of FANCA at serine residue 1149. This information is crucial for proper interpretation of experimental results, as it helps distinguish the specific band of interest from potential non-specific signals or cross-reactive proteins.

What species reactivity has been confirmed for this antibody?

The Phospho-FANCA (Ser1149) Antibody demonstrates confirmed reactivity with human, mouse, and rat samples . This cross-species reactivity makes the antibody valuable for comparative studies across different model organisms. Based on sequence homology analysis, the antibody is also predicted to react with bovine, horse, and sheep samples, though these predicted reactivities would require experimental validation before use in critical experiments . The broad species reactivity profile enables researchers to conduct evolutionary studies or to translate findings between rodent models and human systems when investigating FANCA function and phosphorylation dynamics.

What is the significance of FANCA phosphorylation at Ser1149 in biological systems?

FANCA phosphorylation at Ser1149 represents an important post-translational modification that likely regulates protein function in the Fanconi anemia pathway. FANCA is a critical component of the Fanconi anemia complementation group, which plays essential roles in DNA repair mechanisms, particularly in response to DNA crosslinking agents . Phosphorylation at specific serine residues, including Ser1149, may modulate FANCA's ability to interact with other proteins in the complex, its subcellular localization, or its activation status. Mutations in FANCA gene are the most common cause of Fanconi anemia, a genetically heterogeneous recessive disorder characterized by cytogenetic instability, hypersensitivity to DNA crosslinking agents, increased chromosomal breakage, and defective DNA repair . Studying this specific phosphorylation event can provide insights into the regulation of DNA repair pathways and potential therapeutic targets for Fanconi anemia and related conditions.

How can I validate the specificity of Phospho-FANCA (Ser1149) Antibody in my experimental system?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For Phospho-FANCA (Ser1149) Antibody, a multi-faceted approach is recommended. First, conduct a phosphatase treatment control experiment where you divide your sample into two portions - treat one with lambda phosphatase to remove phosphate groups and leave the other untreated. The antibody signal should significantly decrease or disappear in the phosphatase-treated sample when compared to the untreated control in Western blot analysis. Second, use FANCA-deficient cell lines (either natural mutants or CRISPR/Cas9-generated knockouts) as negative controls to confirm absence of signal. Third, employ peptide competition assays using both phosphorylated and non-phosphorylated peptides containing the Ser1149 sequence - only the phosphorylated peptide should block antibody binding. Finally, validate results using an orthogonal method such as mass spectrometry to confirm phosphorylation at Ser1149 . This comprehensive validation strategy ensures that observed signals genuinely represent phosphorylated FANCA at Ser1149.

What are the optimal conditions for using Phospho-FANCA (Ser1149) Antibody in Western blotting?

For optimal Western blotting results with Phospho-FANCA (Ser1149) Antibody, consider the following methodological approach: First, ensure complete protein extraction by using a lysis buffer containing phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, and β-glycerophosphate) to preserve phosphorylation status. Use fresh samples whenever possible, as freeze-thaw cycles can degrade phosphorylated epitopes. For electrophoresis, load 20-40 μg of total protein per lane on a 6-8% SDS-PAGE gel to optimize separation of high molecular weight proteins like FANCA (163 kDa). After transfer to nitrocellulose or PVDF membrane, block using 5% BSA in TBST rather than milk, as milk contains phosphoproteins that may interfere with phospho-specific antibody binding. Dilute the Phospho-FANCA (Ser1149) Antibody at 1:500-1:2000 in blocking buffer and incubate overnight at 4°C for best results . For detection, both chemiluminescence and fluorescence-based systems work well, but fluorescence may offer better quantitative analysis. Include positive controls such as K562 cell lysates, which have been validated to express phosphorylated FANCA . Remember to strip and reprobe with total FANCA antibody to calculate the phosphorylation/total protein ratio for accurate interpretation.

What experimental conditions can induce or modulate FANCA phosphorylation at Ser1149?

FANCA phosphorylation at Ser1149 can be experimentally modulated through several approaches to study its regulation and functional significance. DNA damaging agents, particularly DNA crosslinking agents like mitomycin C (MMC) and cisplatin, are effective inducers of the Fanconi anemia pathway activation and likely increase FANCA phosphorylation. Treatment with 40-100 ng/ml MMC for 16-24 hours typically activates the pathway. Additionally, ionizing radiation (2-10 Gy) and ultraviolet light (UV) exposure can stimulate DNA damage response pathways that may affect FANCA phosphorylation status. Cell cycle synchronization experiments are valuable for understanding temporal regulation, as FANCA phosphorylation may vary throughout cell cycle phases, with potential enrichment during S phase when DNA replication occurs. For studying regulatory kinases, specific inhibitors of ATM/ATR kinases (such as KU-55933 or VE-821) can help determine if these DNA damage response kinases are involved in Ser1149 phosphorylation. Hypoxic conditions (1% O₂) may also modulate FANCA pathway activation, as have been shown for other DNA repair mechanisms. When designing these experiments, include appropriate time course analyses (typically 0-24 hours) to capture both rapid and delayed phosphorylation events, and analyze both nuclear and cytoplasmic fractions separately, as phosphorylation may influence protein localization.

How does phosphorylation at Ser1149 compare with other known FANCA phosphorylation sites?

FANCA undergoes multiple phosphorylation events beyond Ser1149, with distinct functional implications for each site. While Ser1149 phosphorylation appears to be involved in regulating FANCA's function in DNA repair pathways, it represents just one regulatory node in a complex network. Another significant phosphorylation site is Ser1449, for which specific antibodies are also available . The phosphorylation at these different sites may occur sequentially or in response to different cellular stimuli, potentially creating a phosphorylation code that dictates FANCA activity and interactions. Comparative studies using antibodies against different phosphorylation sites (e.g., using both Phospho-FANCA (Ser1149) and Phospho-FANCA (Ser1449) Antibodies) can reveal whether these modifications occur simultaneously or mutually exclusively, providing insights into regulatory hierarchies. Additionally, mass spectrometry-based phosphoproteomic analysis has identified other potential phosphorylation sites on FANCA, though their functional significance remains less characterized than Ser1149. To comprehensively understand FANCA regulation, researchers should consider designing experiments that monitor multiple phosphorylation sites simultaneously using a combination of phospho-specific antibodies and phosphoproteomic approaches.

What are the recommended immunohistochemistry protocols for Phospho-FANCA (Ser1149) Antibody?

For optimal immunohistochemistry results using Phospho-FANCA (Ser1149) Antibody, the following methodology is recommended: Begin with either paraffin-embedded or frozen tissue sections, with paraffin sections requiring antigen retrieval to unmask epitopes. For antigen retrieval, use citrate buffer (pH 6.0) and heat treatment (95-100°C for 15-20 minutes) followed by cooling to room temperature. When using formalin-fixed tissues, note that extended fixation can mask phospho-epitopes, so fixation time should be optimized and standardized. Block endogenous peroxidase activity with 3% hydrogen peroxide in methanol for 10 minutes, then block non-specific binding with 5% normal goat serum in PBS containing 0.1% Triton X-100 for 1 hour at room temperature. Apply Phospho-FANCA (Ser1149) Antibody at a dilution of 1:100-1:300 in blocking buffer and incubate overnight at 4°C in a humidified chamber . For detection, use an appropriate secondary antibody system such as biotin-streptavidin-HRP or polymer-based detection systems, followed by DAB (3,3′-diaminobenzidine) chromogen development. Counterstain with hematoxylin, dehydrate, clear, and mount with permanent mounting medium. Always include both positive controls (tissues known to express phosphorylated FANCA) and negative controls (either primary antibody omission or phosphatase-treated sections) to validate staining specificity.

How can phosphorylated FANCA be effectively studied in combination with other Fanconi anemia proteins?

To study phosphorylated FANCA in relation to other Fanconi anemia proteins, researchers should employ multiplexed approaches that preserve protein interactions and localization. Co-immunoprecipitation (Co-IP) experiments using Phospho-FANCA (Ser1149) Antibody can pull down phosphorylated FANCA along with its interacting partners for subsequent analysis by Western blot or mass spectrometry. This approach reveals how phosphorylation impacts FANCA's incorporation into the Fanconi anemia core complex with partners like FANCB, FANCC, FANCE, FANCF, and FANCG. For multiplexed imaging studies, combine the Phospho-FANCA (Ser1149) Antibody with antibodies against other Fanconi anemia proteins using multi-color immunofluorescence, ensuring antibodies are raised in different host species to avoid cross-reactivity. Proximity ligation assays (PLA) offer enhanced sensitivity for detecting protein-protein interactions between phosphorylated FANCA and other complex members within intact cells at endogenous expression levels. For temporal dynamics, live-cell imaging using split fluorescent protein systems or FRET-based reporters can track how phosphorylation affects complex assembly in real-time. Complementary biochemical techniques such as sucrose gradient ultracentrifugation or size exclusion chromatography can separate intact complexes containing phosphorylated FANCA from free proteins. When designing these experiments, consider that phosphorylation status may change rapidly upon cellular stress, requiring careful timing and potentially rapid sample processing to preserve physiologically relevant modifications.

What techniques can be used to investigate the functional consequences of FANCA phosphorylation at Ser1149?

To investigate the functional impact of FANCA phosphorylation at Ser1149, researchers should employ a multi-faceted approach combining genetic, biochemical, and cellular techniques. Begin with site-directed mutagenesis to generate phospho-mimetic (S1149D or S1149E) and phospho-deficient (S1149A) FANCA mutants for expression in FANCA-deficient cell lines. These complementation experiments allow direct comparison of how phosphorylation status affects FANCA function. Assess DNA repair efficiency using standard assays such as chromosome breakage analysis after mitomycin C treatment, DNA repair foci formation (monitoring γH2AX, RAD51, or FANCD2 foci), and comet assays to measure DNA strand breaks. For protein interaction studies, compare wild-type and mutant FANCA using immunoprecipitation followed by mass spectrometry to identify phosphorylation-dependent binding partners. Chromatin immunoprecipitation (ChIP) can reveal if Ser1149 phosphorylation affects FANCA recruitment to damaged DNA. Cell cycle analysis using flow cytometry will determine if phosphorylation status impacts cell cycle progression or checkpoints. For in vivo relevance, generate knock-in mouse models expressing the phospho-mutants and assess phenotypes related to Fanconi anemia. Finally, use structural biology approaches such as hydrogen-deuterium exchange mass spectrometry (HDX-MS) to understand how phosphorylation induces conformational changes in FANCA. This comprehensive strategy will elucidate both the molecular mechanisms and cellular outcomes of Ser1149 phosphorylation.

How can I troubleshoot weak or non-specific signals when using Phospho-FANCA (Ser1149) Antibody?

When encountering weak or non-specific signals with Phospho-FANCA (Ser1149) Antibody, implement the following troubleshooting strategy: For weak signals, first verify phosphorylation status by treating positive control cells with DNA crosslinking agents like mitomycin C to enhance FANCA pathway activation. Ensure complete preservation of phospho-epitopes by using fresh phosphatase inhibitor cocktails in all buffers and avoiding repeated freeze-thaw cycles. Try increasing antibody concentration (up to 1:250 dilution) or extending incubation time to overnight at 4°C. For non-specific bands in Western blotting, optimize blocking conditions by using 5% BSA instead of milk and increasing blocking time to 2 hours at room temperature. Consider using alternative membranes (PVDF may retain phosphoproteins better than nitrocellulose for some applications) and optimize transfer conditions for high molecular weight proteins (163 kDa) by using lower methanol concentration or adding SDS to transfer buffer. For background issues in immunostaining, implement more stringent washing steps (4-5 washes of 10 minutes each), use 0.3% Triton X-100 in wash buffers to reduce hydrophobic interactions, and pre-absorb antibody with non-specific proteins. If high background persists, titrate secondary antibody and consider using highly cross-adsorbed secondary antibodies to minimize species cross-reactivity. For all applications, compare results with a total FANCA antibody to confirm protein expression and loading. If necessary, immunodeplete the antibody with non-phosphorylated peptide to enhance phospho-specificity.

What are the detailed specifications of commercially available Phospho-FANCA (Ser1149) Antibodies?

The following table summarizes the technical specifications of commercially available Phospho-FANCA (Ser1149) Antibodies based on manufacturer data:

This comprehensive specification table provides researchers with essential information for experimental planning, including appropriate dilutions for different applications and expected molecular weight for proper band identification .

What controls should be included when performing experiments with Phospho-FANCA (Ser1149) Antibody?

A robust experimental design with appropriate controls is essential when working with Phospho-FANCA (Ser1149) Antibody. The following table outlines recommended controls for different experimental applications:

Implementing these controls provides confidence in experimental results and helps troubleshoot issues that may arise during experiments with Phospho-FANCA (Ser1149) Antibody .

How does FANCA phosphorylation fit within the broader Fanconi anemia pathway?

FANCA phosphorylation represents a critical regulatory mechanism within the complex Fanconi anemia (FA) pathway, which orchestrates DNA interstrand crosslink (ICL) repair. The following diagram outlines the position of phosphorylated FANCA within this pathway:

Fanconi Anemia Pathway Components and Interactions:

• DNA Damage Recognition: ICLs detected during DNA replication

• FA Core Complex Assembly: Includes FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM

• FANCA Phosphorylation: Occurs at multiple sites including Ser1149 and Ser1449

• E3 Ubiquitin Ligase Activity: Phosphorylated FANCA contributes to core complex functionality

• FANCD2/FANCI Monoubiquitination: Key step activated by functional core complex

• Recruitment of DNA Repair Factors: Including FANCD1(BRCA2), FANCJ, FANCN, and FANCO

• DNA Repair: Homologous recombination and translesion synthesis

FANCA phosphorylation at Ser1149 likely influences core complex assembly, stability, or activation. When FANCA is properly phosphorylated, the core complex functions effectively to monoubiquitinate FANCD2/FANCI, allowing proper DNA repair. Disruption of this phosphorylation through mutations or regulatory defects may contribute to FA pathogenesis by impairing the DNA damage response. This positioning of FANCA phosphorylation within the larger pathway explains why studying this specific modification is crucial for understanding both normal DNA repair mechanisms and the molecular basis of Fanconi anemia .

What is the relevance of FANCA phosphorylation studies to understanding Fanconi anemia pathogenesis?

FANCA phosphorylation studies provide critical insights into the molecular mechanisms underlying Fanconi anemia pathogenesis. Fanconi anemia is predominantly caused by mutations in the FANCA gene, accounting for approximately 60-65% of all cases . Phosphorylation at Ser1149 and other sites likely serves as a regulatory switch that controls FANCA function within the Fanconi anemia core complex. When FANCA phosphorylation is disrupted—either through direct mutations at phosphorylation sites, mutations that alter protein conformation preventing kinase access, or defects in upstream signaling pathways—the entire DNA repair mechanism becomes compromised. This results in the cellular hallmarks of Fanconi anemia: chromosomal instability, hypersensitivity to DNA crosslinking agents, and defective DNA repair. Research using Phospho-FANCA (Ser1149) Antibody can help classify patient mutations based on their effects on phosphorylation status, potentially creating functional categories that correlate with disease severity or treatment response. Understanding the detailed phosphorylation-dependent regulation of FANCA may also reveal points of therapeutic intervention, where small molecules could potentially stabilize protein interactions normally facilitated by phosphorylation, bypassing genetic defects in some patients.

How can Phospho-FANCA (Ser1149) Antibody be used in cancer research beyond Fanconi anemia?

Phospho-FANCA (Ser1149) Antibody offers valuable applications in cancer research beyond Fanconi anemia studies. The Fanconi anemia pathway intersects with BRCA-mediated DNA repair mechanisms, making it relevant to multiple cancer types, particularly those with DNA repair deficiencies. In breast and ovarian cancers, where BRCA1/2 mutations are prevalent, studying FANCA phosphorylation can reveal compensatory DNA repair mechanisms that might explain treatment resistance. For example, researchers can use the antibody to assess whether hyperphosphorylation of FANCA correlates with platinum drug resistance in ovarian cancer cell lines or patient samples. In high-throughput drug screening approaches, this antibody can serve as a pharmacodynamic marker to identify compounds that modulate the Fanconi anemia pathway, potentially discovering synthetic lethal interactions in cancer cells with specific genetic backgrounds. Additionally, immunohistochemistry with Phospho-FANCA (Ser1149) Antibody on cancer tissue microarrays can reveal patterns of pathway activation across different tumor types and stages, potentially identifying patient subgroups that might benefit from specific targeted therapies. The antibody is also valuable for investigating how cancer-associated mutations in genes encoding kinases or phosphatases might dysregulate FANCA phosphorylation, contributing to genomic instability—a hallmark of cancer progression.