RAD51D Antibody

What is RAD51D and why is it important in research?

RAD51D is a RAD51 paralog that plays a crucial role in the homologous recombination repair (HRR) pathway of double-stranded DNA breaks. It forms part of the RAD51 paralog protein complex BCDX2 which acts in the BRCA1-BRCA2-dependent HR pathway. Upon DNA damage, the BCDX2 complex acts downstream of BRCA2 recruitment and upstream of RAD51 recruitment . RAD51D binds to single-stranded DNA and possesses DNA-dependent ATPase activity. Its importance in research stems from its involvement in cancer development, particularly in breast and ovarian cancer, and its role in determining sensitivity to PARP inhibitor therapies .

When selecting a RAD51D antibody, researchers should consider:

• Antibody type: Monoclonal antibodies like Rad51D 5B3/6 and Rad51D 5A8/4 offer high specificity, while polyclonal antibodies may provide higher sensitivity but potentially more background .

• Host species: Available in mouse (monoclonal) and rabbit (polyclonal) variants .

• Target species reactivity: Confirm reactivity with your sample species (human, mouse, hamster) .

• Applications: Ensure the antibody is validated for your intended application (WB, IP, ICC/IF, IHC-P) .

• Molecular weight detection: RAD51D has a calculated molecular weight of 35 kDa, but is typically observed at 30-40 kDa in Western blots .

• Storage conditions: Most RAD51D antibodies require storage at -15° C to -25° C in PBS buffer with preservatives .

How can I optimize RAD51D antibody detection in Western blot applications?

For optimal Western blot detection of RAD51D:

• Sample preparation: Use appropriate lysis buffers that preserve protein integrity while efficiently extracting nuclear proteins.

• Protein loading: Load 15-20 μg of total protein per lane, as demonstrated in validation studies .

• Antibody dilution: Use recommended dilutions (for example, 1:1000-1:6000 for antibody 30068-1-AP) .

• Controls: Include both positive controls (RAD51D-expressing cells like HEK-293, HeLa, or A2780) and negative controls (RAD51D knockdown cells) .

• Expected band size: Look for bands in the 30-40 kDa range, as the calculated molecular weight is 35 kDa .

• Validation strategy: Consider using RAD51D transfected cell lysates alongside non-transfected controls to confirm specificity, as shown in validation studies where both transfected and non-transfected 293T cell lysates were compared .

What are the recommended protocols for studying RAD51D foci formation in DNA damage response?

To study RAD51D foci formation in response to DNA damage:

• Experimental setup:

  • Treat cells with DNA-damaging agents (such as those used in RAD51 and γ-H2AX foci formation assays) .

  • Include wildtype cells, cells with primary RAD51D mutations, and cells with secondary mutations for comparison .

• Immunofluorescence protocol:

  • Fix cells appropriately (commonly with 4% paraformaldehyde).

  • Permeabilize cells to allow antibody access to nuclear proteins.

  • Block with appropriate buffer to reduce non-specific binding.

  • Incubate with RAD51D antibody (ab168463 has been validated for ICC/IF) .

  • Use appropriate fluorescently-labeled secondary antibody.

  • Co-stain with DAPI to visualize nuclei.

• Analysis methods:

  • Quantify foci formation using fluorescence microscopy.

  • Count the number of foci per nucleus (typically >5 foci per nucleus is considered positive).

  • Analyze at least 100 cells per condition for statistical significance.

  • Perform statistical analysis using appropriate tests like ANOVA followed by the Tukey test .

Research has demonstrated that RAD51 and γ-H2AX foci formation assays can effectively illustrate deficient HR repair in tumor samples with primary RAD51D mutations compared to those with secondary mutations .

How can RAD51D antibodies be used to investigate PARP inhibitor resistance mechanisms?

RAD51D antibodies are valuable tools for investigating PARP inhibitor resistance mechanisms through:

• Expression analysis:

  • Use Western blot and immunohistochemistry to assess RAD51D expression levels before and after developing resistance to PARP inhibitors .

  • Research has shown that RAD51D expression can be restored in resistant tumors through secondary mutations that correct the reading frame of primary mutations .

• Functional assays:

  • Conduct γ-H2AX foci formation assays to assess HR repair proficiency, which correlates with PARP inhibitor resistance .

  • Perform cell viability assays with PARP inhibitors (olaparib, niraparib) to correlate RAD51D status with drug sensitivity .

• Mutation analysis workflow:

  • Generate cell lines expressing wildtype RAD51D, primary mutations, or secondary mutations.

  • Validate expression status by Western blotting.

  • Assess PARP inhibitor sensitivity across these cell lines.

  • Correlate with RAD51D expression and function.

In a clinical case study, immunohistochemical analysis demonstrated that a germline RAD51D frameshift mutation resulted in low RAD51D expression in initial tumor samples (PARP inhibitor sensitive), while a secondary deletion mutation restored RAD51D expression in resistant tumors, as confirmed by increased IHC staining .

What cell line models are recommended for RAD51D antibody validation?

Based on published research, the following cell lines are recommended for RAD51D antibody validation:

When validating antibodies, it's recommended to:

• Include both high and low expressing cell lines

• Test RAD51D knockdown controls using shRNA constructs for specificity confirmation

• Compare antibody performance in multiple applications (WB, IF, IP) when possible

How can I generate RAD51D expression constructs for functional studies?

Based on published methodologies, researchers can generate RAD51D expression constructs following these steps:

• Obtain cDNA source:

  • Commercial sources like Vigene Biosciences (RAD51D, #CH822036)

  • Public repositories or plasmid banks

• Cloning strategy:

  • Subclone RAD51D cDNA into appropriate expression vectors (e.g., lentiviral vector pLVX-IRES-Neo)

  • Use high-fidelity polymerases such as PrimeSTAR Max DNA Polymerase for amplification

  • Employ efficient cloning kits like ClonExpress II One Step Cloning Kit

• Mutation generation:

  • Introduce specific mutations using PCR-based mutagenesis with KOD One PCR Master Mix or similar

  • For frameshift mutations like K91fs (c.271_272insTA), design appropriate primers

  • Verify all constructs by DNA sequencing

• Viral packaging and transduction:

  • Package lentiviral constructs with appropriate packaging plasmids (psPAX2 and pMD2.G)

  • Harvest viral particles after 48 hours

  • Filter through 0.45-μm filters before transduction

  • Add polybrene (8 mg/mL) during infection

  • Select stable cells using appropriate antibiotics (puromycin at 2 mg/mL or G418 at 5 mg/mL) for 1-2 weeks

• Validation:

  • Confirm expression by Western blot using validated RAD51D antibodies

  • Verify function using appropriate assays (e.g., HR repair assays, PARP inhibitor sensitivity)

How can RAD51D antibodies be used to evaluate patient samples in cancer research?

RAD51D antibodies can be valuable tools for evaluating patient samples in cancer research:

• Immunohistochemical analysis:

  • RAD51D antibodies can be used to assess expression levels in tumor samples from cancer patients .

  • Different expression patterns have been observed between tumors with functional versus non-functional RAD51D due to mutations .

  • This can help identify patients who might benefit from PARP inhibitor therapy or explain resistance mechanisms.

• Mutation-specific analysis:

  • Researchers have used IHC with RAD51D antibodies to demonstrate how different mutations affect protein expression .

  • In one study, low IHC staining of RAD51D was observed in tumor sections with germline frameshift mutations, while restored staining was seen in samples with secondary compensatory mutations .

• Correlation with treatment response:

  • RAD51D expression status determined by IHC has been correlated with response to PARP inhibitor therapy .

  • The presence of RAD51D protein correlates with homologous recombination proficiency and potential PARP inhibitor resistance .

• Sample preparation recommendations:

  • Use appropriate fixation methods compatible with the antibody

  • Include positive and negative control tissues

  • Consider dual staining with other HR pathway proteins for context

What methodologies are recommended for detecting RAD51D mutations and correlating with antibody staining patterns?

For comprehensive RAD51D mutation detection and correlation with antibody staining:

• Genetic screening approaches:

  • Next-generation sequencing (NGS) of RAD51D genes

  • Targeted panel testing for hereditary cancer genes

  • Whole exome sequencing for discovery of novel variants

• Correlation methodology:

  • Perform immunohistochemistry using validated RAD51D antibodies on tumor sections

  • Quantify staining intensity and pattern

  • Correlate with specific genetic alterations

  • Document changes in expression patterns before and after treatment

• Functional validation:

  • Generate cell models expressing specific RAD51D variants

  • Assess protein expression by Western blot

  • Perform functional assays (RAD51 and γ-H2AX foci formation)

  • Test sensitivity to PARP inhibitors

• Clinical correlation:

  • Monitor treatment response in patients with various RAD51D mutations

  • Use liquid biopsy and ctDNA analysis to detect emerging resistant mutations

  • Correlate antibody staining patterns with clinical outcomes

Research has shown that germline RAD51D mutations, particularly frameshift variants like K91fs, can lead to loss of protein expression detectable by antibody staining, while secondary mutations can restore expression and function, leading to treatment resistance .

What are common issues encountered with RAD51D antibodies and how can they be resolved?

Common issues with RAD51D antibodies and their solutions include:

• Low or no signal in Western blot:

  • Increase antibody concentration (try 1:1000 before moving to higher concentrations)

  • Optimize protein extraction methods to ensure nuclear proteins are efficiently extracted

  • Use fresh samples and avoid repeated freeze-thaw cycles

  • Verify sample integrity with loading controls

  • Check if your cell line expresses RAD51D (use positive control cell lines like HEK-293, HeLa, or A2780)

• High background:

  • Increase blocking time or concentration

  • Use more stringent washing conditions

  • Reduce primary and secondary antibody concentrations

  • Use highly cross-adsorbed secondary antibodies

  • Consider alternative blocking agents (BSA vs. milk)

• Non-specific bands:

  • Verify the expected molecular weight (30-40 kDa for RAD51D)

  • Use RAD51D knockout or knockdown controls

  • Increase antibody specificity by pre-adsorption

  • Try different antibody clones or lots

• Poor reproducibility:

  • Standardize protocols and reagents

  • Document lot numbers and storage conditions

  • Aliquot antibodies to avoid repeated freeze-thaw cycles

  • Consider using automated systems where possible

• Weak immunofluorescence signal:

  • Optimize fixation and permeabilization methods

  • Increase antibody concentration or incubation time

  • Use signal amplification methods

  • Adjust image acquisition settings

What controls should be included when using RAD51D antibodies for various applications?

Proper experimental controls are essential for reliable results with RAD51D antibodies:

Additionally, for mutation studies, include:

• Wildtype RAD51D expressing cells

• Cells expressing known RAD51D mutations (e.g., K91fs)

• Cells with characterized secondary/reversion mutations

How are RAD51D antibodies being used in novel research on PARP inhibitor resistance mechanisms?

Recent advances in using RAD51D antibodies to study PARP inhibitor resistance include:

• Reversion mutation detection:

  • Researchers have identified a novel reversion mutation involving a 12 bp deletion (LRG_516t1:c.271_282del) that can eliminate the frameshift effect of a germline 2 bp duplication in RAD51D .

  • RAD51D antibodies were crucial in demonstrating restored protein expression in resistant tumors through immunohistochemical analysis .

• Functional validation approaches:

  • RAD51D antibodies are being used in combination with RAD51 and γ-H2AX foci formation assays to assess homologous recombination repair capacity .

  • These assays have demonstrated that deficient HR repair in initial tumor samples can be complemented by secondary RAD51D mutations .

• Temporal monitoring of resistance:

  • Researchers are applying RAD51D antibodies to study the evolution of resistance by comparing expression in samples collected before treatment, during response, and at progression .

  • This approach has revealed how RAD51D expression changes correlate with clinical response and resistance.

• Liquid biopsy correlation:

  • Studies have shown that reversion mutations can be detected in circulating tumor DNA (ctDNA) .

  • This allows for non-invasive monitoring of potential resistance mechanisms using RAD51D antibodies on cell-free DNA.

• Combination therapy studies:

  • RAD51D antibodies are being used to identify tumors that might benefit from combination therapies designed to overcome resistance mechanisms.

  • Expression patterns can help predict which patients might develop resistance and guide preemptive therapeutic strategies.

What emerging techniques are enhancing the utility of RAD51D antibodies in cancer research?

Emerging techniques enhancing RAD51D antibody utility include:

• Multiplex immunofluorescence:

  • Allows simultaneous detection of RAD51D with other HR pathway proteins (BRCA1/2, RAD51, etc.)

  • Provides context for RAD51D expression within the complete DNA repair pathway

  • Enables better characterization of homologous recombination deficiency phenotypes

• Single-cell analysis:

  • Application of RAD51D antibodies in single-cell protein analysis technologies

  • Reveals heterogeneity in expression and function within tumor populations

  • May explain why some cells survive PARP inhibitor therapy while others don't

• Proximity ligation assays (PLA):

  • Detect protein-protein interactions involving RAD51D in situ

  • Provide spatial information about RAD51D interactions with other HR components

  • Enable visualization of complex formation in response to DNA damage

• Live-cell imaging:

  • Development of antibody-based fluorescent reporters for RAD51D

  • Allows real-time monitoring of RAD51D recruitment to DNA damage sites

  • Provides temporal information about HR pathway activation

• AI-assisted image analysis:

  • Machine learning algorithms for automated quantification of RAD51D foci

  • Improves consistency and throughput in foci counting experiments

  • Enables detection of subtle patterns that might escape manual analysis

• CRISPR-engineered cellular models:

  • Creation of endogenously tagged RAD51D to avoid overexpression artifacts

  • Generation of allelic series of RAD51D mutations for systematic functional studies

  • Development of isogenic cell lines with specific RAD51D variants for drug screening