Phospho-FANCG (Ser383) Antibody

What is FANCG and what is the significance of its phosphorylation at Ser383?

FANCG (Fanconi anemia complementation group G) is a DNA repair protein also known as XRCC9 (X-ray repair complementing defective repair in Chinese hamster cells 9). It functions as a critical component of the Fanconi anemia pathway, which is essential for DNA interstrand cross-link repair and the maintenance of normal chromosome stability .

Ser383 phosphorylation of FANCG occurs specifically during mitosis and is functionally important for the proper operation of the Fanconi anemia repair pathway . Research has demonstrated that mutation of Ser383 to alanine abolishes the phosphorylation of FANCG at mitosis and impairs the ability to correct FA-G mutant cells, highlighting the essential nature of this post-translational modification .

The phosphorylation sequence context around Ser383 is "RFsPP" (where "s" represents the phosphorylated serine) , which enables the generation of highly specific antibodies that recognize only the phosphorylated form of the protein.

What is the expression pattern and cellular localization of FANCG?

FANCG shows tissue-specific expression patterns, with high expression observed in:

• Testis

• Thymus

• Lymphoblasts

At the subcellular level, FANCG exhibits a dual localization pattern:

This differential localization is relevant to researchers studying FANCG function, as the protein's nuclear localization is associated with its role in DNA repair processes .

What are the key specifications of available Phospho-FANCG (Ser383) antibodies?

Most commercially available Phospho-FANCG (Ser383) antibodies share similar specifications:

These antibodies are specifically designed to detect endogenous levels of FANCG protein only when phosphorylated at Ser383, making them valuable tools for studying the phosphorylation state of FANCG in various biological contexts .

What research applications are Phospho-FANCG (Ser383) antibodies best suited for?

Phospho-FANCG (Ser383) antibodies have been validated for several key applications:

• Western Blot (WB): The most common application, typically at dilutions of 1:500-1:2000, for detecting phosphorylated FANCG in protein extracts .

• Enzyme-Linked Immunosorbent Assay (ELISA): Used at dilutions around 1:40000 for quantitative detection of phosphorylated FANCG .

• Immunohistochemistry (IHC): Some antibodies have been validated for IHC applications on paraffin sections, enabling visualization of phosphorylated FANCG in tissue samples .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Some versions are suitable for cellular localization studies at dilutions of 1:50-1:200 .

When selecting an application, researchers should consider the specific validation data provided by the manufacturer for their antibody of interest.

How should researchers design experiments to study FANCG phosphorylation during the cell cycle?

To effectively study FANCG phosphorylation during the cell cycle, particularly during mitosis when Ser383 phosphorylation is most pronounced , researchers should design experiments that:

• Synchronize cell populations: Use methods such as double thymidine block, nocodazole treatment, or selective detachment of mitotic cells to obtain enriched populations at specific cell cycle phases.

• Generate proper controls: Include:

  • Asynchronous cell populations

  • Cells arrested at different cell cycle phases

  • Phosphatase-treated samples to confirm phospho-specificity

  • Peptide competition controls using phospho-peptide (RFsPP) and non-phospho-peptide

• Validate phosphorylation status: Consider complementary approaches such as:

  • Phospho-specific antibodies to both Ser383 and Ser387 sites (dual phosphorylation)

  • Phosphatase treatments of lysates

  • Site-directed mutagenesis (S383A) for functional validation

• Monitor cell cycle markers: Include analysis of established cell cycle phase markers (e.g., Cyclin B1 for G2/M) to correlate FANCG phosphorylation with specific cell cycle phases.

Research has shown that cdc2 (CDK1) is likely the kinase responsible for phosphorylating FANCG at Ser383 during mitosis , so researchers might also consider including CDK1 inhibitors or knockdown approaches in their experimental design.

What are the optimal protocols for using Phospho-FANCG (Ser383) antibody in Western blot applications?

For optimal Western blot results with Phospho-FANCG (Ser383) antibody:

Sample Preparation:

• Lyse cells in a buffer containing phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, phosphatase inhibitor cocktail)

• Use RIPA or NP-40 based lysis buffers for total protein extraction

• Include protease inhibitors to prevent degradation

• For enriched nuclear extracts, consider using nuclear/cytoplasmic fractionation protocols

Western Blot Protocol:

• Load 20-50 μg of total protein per lane

• Use 8-10% SDS-PAGE gels (optimal for 69 kDa proteins)

• Transfer to PVDF or nitrocellulose membranes

• Block with 5% BSA in TBST (not milk, as it contains phosphatases)

• Incubate with Phospho-FANCG (Ser383) antibody at 1:500-1:2000 dilution overnight at 4°C

• Wash thoroughly with TBST

• Use appropriate HRP-conjugated secondary antibody (anti-rabbit IgG)

• Develop using ECL substrate

Validation Controls:

• Include a lane with phosphopeptide competition to confirm specificity

• Consider including phosphatase-treated lysate as a negative control

• Use positive control lysates (e.g., K562 cells or cos-7 cells have been validated)

The typical expected result is a band at approximately 69 kDa representing phosphorylated FANCG protein .

How can Phospho-FANCG (Ser383) antibody be used to investigate FANCG's role in the DNA damage response pathway?

Researchers can leverage Phospho-FANCG (Ser383) antibody to investigate FANCG's role in DNA damage response through several sophisticated approaches:

• Phosphorylation Dynamics After DNA Damage:

  • Treat cells with DNA crosslinking agents (e.g., mitomycin C, cisplatin)

  • Analyze phosphorylation status over a time course

  • Correlate with FANCD2 monoubiquitination, a key downstream event

  • Compare wildtype cells with cells carrying FA pathway mutations

• Chromatin Association Analysis:

  • Perform chromatin fractionation before and after DNA damage

  • Compare phospho-FANCG vs. total FANCG distribution

  • Investigate whether Ser383 phosphorylation affects chromatin binding

• Protein-Protein Interaction Studies:

  • Use phospho-specific antibodies in co-immunoprecipitation experiments

  • Compare interactomes of phosphorylated vs. non-phosphorylated FANCG

  • Investigate whether Ser383 phosphorylation affects complex formation with other FA proteins

• Functional Rescue Experiments:

  • Complement FANCG-deficient cells with wild-type FANCG or S383A mutant

  • Assess DNA repair efficiency using comet assays or chromosome breakage analysis

  • Measure cell survival following exposure to crosslinking agents

Research has already established that FANCG phosphorylation at Ser383 is functionally important, as S383A mutation impairs the ability to correct FA-G mutant cells , suggesting its critical role in the DNA damage response pathway.

What is known about the interrelationship between Ser383 and Ser387 phosphorylation in FANCG?

FANCG undergoes phosphorylation at both Ser383 and Ser387 during mitosis, with both modifications playing important functional roles . The relationship between these phosphorylation events presents an interesting area for advanced research:

• Sequential Phosphorylation: Research suggests that these sites may be phosphorylated in a specific order, potentially creating a phosphorylation cascade. This hypothesis can be tested using phospho-specific antibodies to both sites in time-course experiments.

• Kinase Specificity: While cdc2 (CDK1) appears to be responsible for phosphorylating both sites, the S387A mutation abolished FANCG fusion protein phosphorylation by cdc2 more completely than S383A mutation, suggesting potential differences in kinase efficiency or preference .

• Functional Redundancy vs. Specificity: Both sites are functionally important, but they may serve distinct roles:

  • Both S383A and S387A mutations impaired correction of FA-G mutant cells

  • Researchers can use site-specific antibodies to determine if these phosphorylation events occur independently or coordinately

  • Double mutants (S383A/S387A) could be compared to single mutants to assess synergistic effects

• Structural Implications: The proximity of these phosphorylation sites (S383 and S387) suggests they might cooperatively influence the protein's conformation or interaction capabilities.

Advanced studies could employ phosphomimetic mutations (S→D or S→E) at one site while preventing phosphorylation at the other (S→A) to dissect their individual contributions to FANCG function.

What are common technical challenges when using Phospho-FANCG (Ser383) antibody and how can they be addressed?

Researchers may encounter several challenges when working with Phospho-FANCG (Ser383) antibody:

For definitive validation of signal specificity, researchers should:

• Perform peptide competition assays using the phosphopeptide (RFsPP)

• Include phosphatase-treated lysates as negative controls

• Consider using FANCG knockout or knockdown samples as specificity controls

As demonstrated in published Western blots, authentic phospho-FANCG (Ser383) signal should disappear when competed with the specific phosphopeptide but remain when competed with non-phosphopeptide .

How can researchers differentiate between true biological variations in FANCG phosphorylation and technical artifacts?

Distinguishing biological variations from technical artifacts requires robust experimental design and appropriate controls:

• Biological Replicates vs. Technical Replicates:

  • Perform both biological replicates (independent cell cultures) and technical replicates (multiple analyses of the same sample)

  • Consistent patterns across biological replicates suggest true biological variation

• Internal Controls:

  • Monitor total FANCG levels alongside phospho-FANCG

  • Calculate phospho-FANCG/total FANCG ratios to normalize for expression differences

  • Include housekeeping proteins (e.g., GAPDH, β-actin) for loading control

• Cell Cycle Considerations:

  • Since FANCG Ser383 phosphorylation is cell cycle-dependent (primarily in mitosis), differences in cell cycle distribution can cause apparent variation

  • Include cell cycle markers (e.g., phospho-histone H3) to correlate with phospho-FANCG levels

  • For heterogeneous samples, consider flow cytometry with phospho-FANCG staining to correlate with cell cycle phases

• Cross-Validation Approaches:

  • Use alternative methods to confirm phosphorylation patterns (e.g., mass spectrometry)

  • Employ Phos-tag gels to separate phosphorylated from non-phosphorylated forms

  • Use functional assays that depend on phosphorylation status

• Dose-Response and Time-Course Experiments:

  • True biological variations often follow predictable patterns in response to stimuli

  • Random variations across similar samples suggest technical issues

By implementing these strategies, researchers can confidently attribute observed variations in FANCG phosphorylation to biological phenomena rather than experimental artifacts.

How might phosphorylation at Ser383 functionally interact with other post-translational modifications on FANCG?

The functional interplay between Ser383 phosphorylation and other post-translational modifications (PTMs) on FANCG represents an important frontier in understanding this protein's regulation:

• Phosphorylation Network:

  • Beyond Ser383 and Ser387, FANCG is phosphorylated at Ser7 , suggesting a complex phosphorylation network

  • Research could investigate whether these phosphorylation events occur hierarchically or independently

  • Phosphoproteomics approaches could reveal additional, uncharacterized phosphorylation sites

• Cross-Talk with Other PTM Types:

  • While phosphorylation has been well-studied, FANCG may undergo other modifications (ubiquitination, SUMOylation, acetylation)

  • These could be studied using appropriate antibodies and mass spectrometry approaches

  • Of particular interest would be whether Ser383 phosphorylation affects ubiquitination of FANCG or other FA pathway components

• Structural Consequences:

  • Phosphorylation can induce conformational changes that expose or mask domains

  • Structural studies comparing phosphorylated and non-phosphorylated FANCG could reveal how Ser383 phosphorylation affects protein architecture

  • Techniques like hydrogen-deuterium exchange mass spectrometry could identify regions with altered solvent accessibility

• Temporal Dynamics:

  • The timing of different modifications during cell cycle progression or DNA damage response could reveal regulatory mechanisms

  • Single-cell analysis techniques could provide insights into cell-to-cell variation in modification patterns

Understanding these interactions would provide deeper insights into FANCG regulation and potentially reveal new therapeutic targets for Fanconi anemia and related disorders.

What research methodologies are emerging for studying FANCG phosphorylation dynamics in live cells?

Emerging technologies provide exciting opportunities for studying FANCG phosphorylation dynamics with unprecedented spatial and temporal resolution:

• Genetically Encoded Biosensors:

  • Development of FRET-based biosensors that change conformation upon FANCG phosphorylation

  • These could enable real-time visualization of phosphorylation events in living cells

  • Potential designs could incorporate phospho-binding domains that recognize the RFsPP motif

• Phospho-Specific Nanobodies:

  • Engineering of small antibody fragments (nanobodies) specific to phospho-Ser383 FANCG

  • These could be expressed intracellularly as fusion proteins with fluorescent tags

  • Advantages include ability to track endogenous protein without overexpression artifacts

• Optogenetic Approaches:

  • Light-controllable kinases or phosphatases to manipulate FANCG phosphorylation with spatial and temporal precision

  • Could help determine the immediate consequences of phosphorylation/dephosphorylation events

• Super-Resolution Microscopy:

  • Techniques like STORM, PALM, or Expansion Microscopy combined with phospho-specific antibodies

  • Would allow visualization of phospho-FANCG localization at nanoscale resolution

  • Could reveal previously undetectable spatial organization patterns

• Microfluidics and Single-Cell Analysis:

  • Microfluidic platforms for precise delivery of DNA-damaging agents while imaging

  • Single-cell proteomics to quantify phospho-FANCG levels in individual cells

  • Could reveal cell-to-cell heterogeneity in response to damage

These methodologies represent the cutting edge of phosphorylation research and would provide valuable insights into the dynamic regulation of FANCG function in living systems.