RAD51C Antibody

What is RAD51C and why is it significant in research?

RAD51C is a pivotal component of the homologous recombination pathway responsible for repairing DNA double-strand breaks within the nucleus. The nuclear localization of RAD51C is crucial for maintaining genomic stability, preventing mutations, and thwarting neoplastic transformations, thereby underscoring its importance in cellular defense against DNA damage . RAD51C is essential for the activation of checkpoint kinase CHK2 and cell cycle arrest in response to DNA damage, making it a critical factor in both DNA repair and cell cycle regulation . Additionally, RAD51C mutations are associated with Fanconi anemia-like syndrome and have been implicated in breast and ovarian cancer predisposition, highlighting its clinical relevance .

What types of RAD51C antibodies are available for research?

Several types of RAD51C antibodies are available for diverse research applications:

• Mouse monoclonal antibodies (e.g., clone 2H11) - These IgG1 kappa light chain antibodies developed against human RAD51C protein can reliably detect RAD51C from mouse, rat, and human origins .

• Rabbit polyclonal antibodies (e.g., A6961) - These antibodies demonstrate cross-reactivity with human and mouse RAD51C proteins .

• Conjugated antibodies including:

  • Agarose-conjugated (for immunoprecipitation)

  • HRP-conjugated (for enhanced western blot detection)

  • Fluorescently labeled variants (FITC, PE, Alexa Fluor®) for immunofluorescence applications

The choice of antibody depends on specific experimental needs, species compatibility, and detection method requirements.

What are the validated applications for RAD51C antibodies?

RAD51C antibodies have been validated for multiple experimental applications:

RAD51C monoclonal antibody (2H11) shows no cross-reactivity with other members of the RAD51 family such as RAD51B, RAD51D, RAD51, XRCC2, or XRCC3, ensuring specificity in detection .

How should I optimize Western blotting for RAD51C detection?

For optimal Western blot detection of RAD51C:

• Sample preparation: Use appropriate nuclear extraction protocols as RAD51C is predominantly nuclear.

• Expected molecular weight: Look for a band at approximately 42 kDa, which is the calculated molecular weight of RAD51C .

• Antibody dilution: For rabbit polyclonal antibody A6961, use dilutions between 1:500 and 1:2000; adjust based on your specific sample and detection system .

• Secondary antibody: Use HRP-conjugated secondary antibodies specific to your primary antibody species (e.g., HRP Goat Anti-Rabbit IgG for rabbit primaries) .

• Controls: Include extracts from cell lines known to express RAD51C and, if possible, RAD51C-depleted samples as negative controls. Western blot analysis has been successfully performed with extracts from various cell lines using RAD51C antibodies .

• Validation: Confirm specificity by comparing bands before and after RAD51C depletion using siRNA or shRNA approaches .

What is the optimal method for visualizing RAD51C foci formation after DNA damage?

To effectively visualize RAD51C foci formation after DNA damage:

• Cell treatment: Expose cells to ionizing radiation (IR) at doses as low as 1 Gy; RAD51C foci have been successfully detected after irradiation at this threshold .

• Cell lines: Human cell lines such as HeLa, U2OS, WI38, and HCT116 have shown robust RAD51C foci formation after irradiation .

• Immunostaining protocol:

  • Fix cells with an appropriate fixative (typically paraformaldehyde)

  • Permeabilize to allow antibody access

  • Block with suitable agents to prevent non-specific binding

  • Incubate with anti-RAD51C antibody

  • Use fluorescently-labeled secondary antibody or directly-conjugated primary antibody

• Controls: Include non-irradiated controls (which should show minimal or no RAD51C foci) and RAD51C-depleted samples to confirm specificity .

• Alternative approaches: GFP-tagged RAD51C expression can also be used to visualize foci formation in response to damage, which can be useful for confirming antibody specificity .

Research has shown that after irradiation, a significant portion of cells display numerous RAD51C foci, whereas no signal is detected in nonirradiated control cells, demonstrating the specificity of damage-induced foci .

How can I investigate the relationship between RAD51C and other DNA repair proteins?

To study the relationship between RAD51C and other DNA repair proteins:

• Co-localization analysis:

  • Perform dual immunofluorescence staining for RAD51C and other repair proteins

  • Research has shown that GFP-RAD51C accumulates into foci that overlap with RAD51 at sites of DNA damage

• Temporal dynamics:

  • Track the timing of foci formation for RAD51C versus other repair proteins

  • RAD51C has been found to persist at damage sites longer than RAD51, suggesting early and late roles in repair

• Dependency relationships:

  • Deplete RAD51C using siRNA and observe effects on other repair proteins

  • Studies show that RAD51C is required for RAD51 foci formation, but in BRCA2-deficient cells, RAD51C foci form despite the absence of RAD51 foci

• Complex formation analysis:

  • Use immunoprecipitation with RAD51C antibodies to identify interacting partners

  • RAD51C participates in multiple subcomplexes: RAD51B–RAD51C–RAD51D–XRCC2, RAD51B–RAD51C, and RAD51C–XRCC3

This multi-faceted approach can help elucidate RAD51C's role in the complex network of DNA repair pathways.

How can RAD51C antibodies be used to study cell cycle checkpoint functions?

To investigate RAD51C's role in cell cycle checkpoint activation:

• Cell cycle analysis:

  • Transfect cells with RAD51C-specific siRNA or control siRNA

  • Irradiate cells and analyze cell cycle distribution by flow cytometry

  • Research has shown that RAD51C-proficient cells accumulate in S phase after irradiation with 1 Gy, whereas RAD51C-depleted cells fail to do so

• Mitotic entry assessment:

  • Analyze the frequency of mitotic cells positive for phosphorylated histone H3 after irradiation

  • Studies demonstrate that control cells show a reduction in mitotic cells after irradiation, while this arrest is significantly less pronounced in RAD51C-depleted cells

• Checkpoint activation markers:

  • Investigate the activation of checkpoint kinases like CHK2 in the presence and absence of RAD51C

  • Research indicates that RAD51C is required for CHK2 activation in response to DNA damage

• Chromosomal instability analysis:

  • Examine metaphase spreads for chromosomal aberrations in RAD51C-depleted cells

  • Primary mouse embryonic fibroblasts with inhibited RAD51C expression show increased chromatid and chromosome breaks after entering mitosis

These approaches can provide comprehensive insights into RAD51C's checkpoint function, beyond its well-established role in DNA repair.

What role does RAD51C play in ovarian cancer research and how can antibodies facilitate these studies?

RAD51C antibodies are valuable tools in ovarian cancer research:

• Germline variant characterization:

  • RAD51C has been identified in familial and sporadic early-onset ovarian cancer (OC) cases through whole exome sequencing

  • Antibodies can help determine the functional impact of these variants on protein expression and localization

• Loss of heterozygosity (LOH) analysis:

  • LOH analysis of RAD51C loci in OC tumor DNA from variant carriers can be complemented with protein expression studies using antibodies

  • This combined approach helps establish the relationship between genetic alterations and protein expression levels

• Tumor characterization:

  • Immunohistochemistry using RAD51C antibodies can assess protein expression in tumor samples

  • This can help categorize tumors based on DNA repair deficiency status

• Therapeutic response prediction:

  • RAD51C deficiency may sensitize tumors to PARP inhibitors

  • Antibody-based detection of RAD51C can potentially serve as a biomarker for treatment response

• Functional analysis of variants:

  • Express RAD51C variants in cell lines and use antibodies to assess their impact on protein function and localization

  • This helps distinguish between pathogenic and benign variants identified in patient samples

These applications highlight the importance of RAD51C antibodies in translating genetic findings into functional insights relevant to cancer biology and treatment.

How can I confirm the specificity of RAD51C antibody staining?

To verify RAD51C antibody specificity:

• siRNA knockdown validation:

  • Treat cells with RAD51C-specific siRNA and confirm reduced signal

  • Research shows that cells depleted of RAD51C through siRNA treatment are impaired in their ability to form RAD51C foci after irradiation

• Rescue experiments:

  • Express siRNA-resistant RAD51C cDNA constructs in depleted cells

  • Studies have demonstrated that in cells harboring siRNA-resistant RAD51C (RAD51C*), foci formation remains intact despite RAD51C siRNA treatment

• Alternative detection methods:

  • Compare antibody detection with GFP-tagged RAD51C expression

  • Research has validated antibody specificity by showing that stable expression of GFP-tagged RAD51C at levels comparable to endogenous protein produces similar localization patterns

• Cross-reactivity testing:

  • Verify that the antibody does not detect other RAD51 family members

  • The RAD51C monoclonal antibody (2H11) shows no cross-reactivity with RAD51B, RAD51D, RAD51, XRCC2, or XRCC3

These validation steps are crucial for ensuring reliable and interpretable experimental results.

How should I interpret the dual roles of RAD51C in homologous recombination?

When analyzing RAD51C's functions in homologous recombination, consider its proposed dual roles:

• Early role evidence:

  • RAD51C-deficient cell lines lack the ability to form IR-induced RAD51 foci

  • Overexpression of RAD51 can partially rescue the DNA repair defect in RAD51C-defective cells

  • The RAD51B-RAD51C complex functions as a mediator of RAD51 nucleoprotein complex assembly

  • RAD51C interacts directly with RAD51 after DNA damage

• Late role evidence:

  • Association of Holliday junction branch migration and resolution activity with RAD51C

  • Reduced Holliday junction resolution activity in RAD51C-defective cells

  • RAD51C-XRCC3 complex binding to Holliday junctions in vitro

  • Persistence of RAD51C foci at late stages of DNA repair

• Unified model:

  • RAD51C likely acts as a platform for RAD51 filament assembly in early repair stages

  • After RAD51 dissociates, RAD51C persists to recruit proteins for late-stage processes

  • The participation of RAD51C in multiple subcomplexes supports these diverse functions

This interpretation reconciles seemingly contradictory findings and positions RAD51C as a multifunctional component of the homologous recombination pathway.

What emerging applications of RAD51C antibodies should researchers consider?

Researchers should explore these emerging applications of RAD51C antibodies:

• Single-cell analysis:

  • Combine RAD51C antibodies with single-cell technologies to analyze cell-to-cell variation in DNA repair capacity

  • This approach can reveal heterogeneity within populations that bulk assays might miss

• High-throughput screening:

  • Develop antibody-based assays for screening compounds that modulate RAD51C function

  • This could identify novel therapeutics targeting DNA repair pathways

• Liquid biopsy development:

  • Explore RAD51C as a biomarker in circulating tumor cells or extracellular vesicles

  • This could provide minimally invasive methods for monitoring treatment response

• Structural studies:

  • Use antibodies as tools to stabilize RAD51C complexes for structural analysis

  • This could reveal mechanistic insights into RAD51C's diverse functions

• Temporal proteomics:

  • Employ RAD51C antibodies in time-resolved proteomics to capture dynamic changes in protein interactions after DNA damage

  • This would provide a systems-level understanding of RAD51C function

These approaches represent the frontier of RAD51C research and could yield valuable insights into fundamental DNA repair mechanisms and their clinical applications.