RAD9B Antibody

What is RAD9B and why is it significant for antibody-based research?

RAD9B is a gene encoding the Cell cycle checkpoint control protein RAD9B, a crucial component of the DNA damage checkpoint pathway. It plays a vital role in sensing and repairing DNA damage to maintain genomic stability . The protein is an essential component of the 9-1-1 complex and can interact with several checkpoint control proteins including HRAD1, HRAD17, HHUS1, and HHUS1B . RAD9B is particularly significant for research because dysregulation has been linked to genomic instability and cancer development, making it an important target for antibody-based detection in genomics and cancer research . Unlike its paralog RAD9A, RAD9B shows tissue-specific expression, predominantly in testis, suggesting specialized functions that warrant targeted antibody applications .

What are the validated applications for RAD9B antibodies?

RAD9B antibodies have been validated for several experimental applications:

For Western blotting applications, positive detection has been confirmed in mouse brain tissue using a concentration of 6μg/ml, with goat polyclonal to rabbit IgG as secondary antibody . When designing experiments, researchers should note that validation has specifically been performed with human and mouse samples, as these are the species with confirmed reactivity .

How should RAD9B antibodies be stored and handled for optimal performance?

For maximum stability and activity, RAD9B antibodies should be stored according to the following specifications:

• Storage Buffer: 0.03% Proclin 300 as preservative, 50% Glycerol, 0.01M PBS, pH 7.4

• Form: Liquid

• Storage Temperature: Manufacturer recommendations typically suggest -20°C for long-term storage

• Aliquoting: To prevent freeze-thaw cycles, divide into small working aliquots before freezing

When handling the antibody for experiments, ensure all solutions are equilibrated to room temperature before opening. The high purity (>95%, Protein G purified) of commercially available RAD9B antibodies ensures specificity, but validation in your specific experimental system is always recommended .

How does one select the appropriate control samples for RAD9B antibody validation?

When validating RAD9B antibodies for your research, consider these methodological approaches:

• Positive Controls: Mouse brain tissue has been verified as a positive control for Western blotting . For human samples, testicular tissue would be appropriate given RAD9B's predominant expression pattern .

• Negative Controls:

  • Use RAD9B knockdown or knockout cell lines where available

  • Include samples from tissues with minimal RAD9B expression

  • Include a secondary antibody-only control to assess non-specific binding

• Specificity Validation: Compare results with another antibody targeting a different epitope of RAD9B, or use recombinant RAD9B protein as a competitive inhibitor to demonstrate specificity.

Remember that RAD9B expression may vary significantly across tissues, with highest expression in testis and potentially detectable levels in neural tissue as indicated by its role in neural development .

How can RAD9B antibodies be used to investigate neural development disorders?

RAD9B has been implicated in neural tube defects (NTDs), particularly spina bifida, making it a valuable target for neurodevelopmental research . When designing investigations into neural development disorders using RAD9B antibodies, consider these methodological approaches:

• Comparative Protein Expression Analysis: Use RAD9B antibodies in Western blotting to compare expression levels between normal neural tissues and those from individuals with NTDs. Research has shown significant enrichment of rare deleterious RAD9B variants in spina bifida cases compared to controls (8/409 vs. 0/298; p = 0.0241) .

• Subcellular Localization Studies: Use immunofluorescence with RAD9B antibodies to examine the nuclear localization of wild-type versus mutant RAD9B. Previous studies have identified that certain variants (including two frameshift mutants and p.Gln146Glu) affect RAD9B nuclear localization .

• Analysis of Developmental Pathways: Combine RAD9B antibodies with those targeting PAX6 and OCT4 to examine dysregulation of neural differentiation pathways, as RAD9B knockdowns in human embryonic stem cells affected early differentiation through impairing these transcription factors .

• 3D Neural Organoid Investigation: Use RAD9B antibodies to assess protein expression and localization in neural organoid cultures, as RAD9B deficiency has been shown to impede in vitro formation of neural organoids .

When reporting results, correlate antibody-detected RAD9B expression patterns with cell adhesion gene expression, as RNA-seq data has revealed that loss of RAD9B dysregulates cell adhesion genes during organoid formation .

What methodological considerations are important when using RAD9B antibodies to study DNA damage response?

When investigating RAD9B's role in the DNA damage response (DDR) pathway:

• Induction of DNA Damage: Treat cells with DNA-damaging agents (e.g., H₂O₂, gamma radiation) before antibody probing to observe RAD9B's response. Mouse studies have shown that Mrad9b-null embryonic stem cells are hypersensitive to certain DNA-damaging agents compared to Mrad9b+/+ controls .

• Co-immunoprecipitation Studies: Use RAD9B antibodies for co-IP experiments to investigate interactions with other 9-1-1 complex components (RAD1, RAD17, HUS1, HUS1B) under different damage conditions .

• Phosphorylation Analysis: Assess JNK phosphorylation in conjunction with RAD9B detection, as variants p.Ser354Gly, p.Ser10Gly, p.Ile112Met, p.Gln146Glu, and frameshift mutations show decreased ability for activating JNK phosphorylation .

• Cell Cycle Checkpoint Analysis: Monitor G2/M checkpoint control in contexts with altered RAD9B expression, noting that though Mrad9b is essential for embryonic development, Mrad9b-null cells show normal gamma-ray-induced G2/M checkpoint control .

• Differential Response to DNA Damaging Agents: When designing experiments with RAD9B antibodies, consider that RAD9B may mediate resistance to specific DNA damaging agents rather than providing general protection against genomic instability .

How can one investigate the functional consequences of RAD9B variants using antibody-based approaches?

To investigate RAD9B variants using antibody-based approaches:

• Protein Stability Assessment: Use Western blotting with RAD9B antibodies to determine if variants affect protein stability. Previous research demonstrated that frameshift mutants decreased the protein level of RAD9B .

• Nuclear Translocation Assays: Employ cellular fractionation followed by Western blotting or immunofluorescence to assess nuclear localization of RAD9B variants. Studies have shown that certain variants (two frameshift mutants and p.Gln146Glu) affected RAD9B nuclear localization .

• Foci Formation Analysis: Under oxidative stress conditions, assess the ability of variant RAD9B to form DNA damage foci using immunofluorescence. Some variants have failed to translocate to the nucleus and form DNA damage foci under conditions of oxidative stress .

• Proliferation Studies: Compare cell proliferation rates when expressing wild-type versus variant RAD9B. The variant p.Ser354Gly and two frameshift variants have been shown to affect cell proliferation rates .

• Signaling Pathway Analysis: Investigate JNK pathway activation using phospho-specific antibodies alongside RAD9B detection. Multiple variants (p.Ser354Gly, p.Ser10Gly, p.Ile112Met, p.Gln146Glu, and frameshift variants) showed decreased ability for activating JNK phosphorylation .

When designing such experiments, consider using tagged RAD9B constructs alongside antibody detection to distinguish between endogenous and exogenous protein.

What are the key considerations when using RAD9B antibodies in embryonic stem cell research?

When applying RAD9B antibodies in embryonic stem cell research:

• Knockdown Validation: Use RAD9B antibodies to confirm successful knockdown in CRISPR or siRNA experiments. RAD9B knockdowns in human embryonic stem cells have been shown to profoundly affect early differentiation .

• Differentiation Markers: Combine RAD9B antibody detection with markers for OCT4 (pluripotency) and PAX6 (neural ectoderm) to monitor differentiation dynamics. RAD9B knockdowns impair expression of these critical factors .

• Time-course Studies: Perform temporal analysis of RAD9B expression during differentiation protocols to understand its dynamic regulation.

• 3D Organoid Formation: Evaluate RAD9B expression in neural organoid formation, as RAD9B deficiency impedes this process .

• Cell Adhesion Pathway Analysis: Combine detection of RAD9B with cell adhesion proteins, as RNA-seq data revealed that loss of RAD9B dysregulates cell adhesion genes during organoid formation .

• Comparative Analysis: Consider comparing RAD9B with RAD9 (RAD9A) expression, as unlike RAD9A which is expressed in most organs, RAD9B shows tissue-specific expression that may indicate specialized roles in certain lineages .

When interpreting results, remember that RAD9B is essential for embryonic development, as demonstrated in mouse studies, making it a critical factor in early developmental processes that can be monitored using appropriate antibodies .

What are common challenges in RAD9B antibody experiments and how can they be overcome?

When working with RAD9B antibodies, researchers frequently encounter these challenges:

• Low Signal Intensity:

  • Solution: Optimize antibody concentration using a range around the recommended dilutions (WB: 1:500-1:5000; ELISA: 1:2000-1:10000)

  • Approach: Increase incubation time, use signal enhancement systems, or concentrate your protein sample

• Background Issues:

  • Solution: Increase blocking time/concentration and optimize washing steps

  • Approach: Use the specific storage buffer components (0.01M PBS, pH 7.4 with 50% Glycerol) for washing to maintain antibody stability

• Cross-Reactivity:

  • Solution: Validate antibody specificity using RAD9B-deficient cells as negative controls

  • Approach: Consider that RAD9B can co-immunoprecipitate with RAD9 (RAD9A) , so interpretation must account for potential interaction effects

• Variable Expression Levels:

  • Solution: Remember that RAD9B is predominantly expressed in testis , so detection in other tissues may require sensitive methods

  • Approach: Use positive controls from tissues with confirmed expression (e.g., mouse brain for WB)

• Nuclear Localization Assessment:

  • Solution: For studying RAD9B variants that affect nuclear localization, optimize your fractionation or immunofluorescence protocol

  • Approach: Include controls for known variants (e.g., frameshift mutants or p.Gln146Glu) that affect localization

How can RNA-seq and immunoblotting data be integrated to study RAD9B function in development?

To effectively integrate RNA-seq and immunoblotting approaches when studying RAD9B:

• Targeted Pathway Validation: RNA-seq studies indicate that RAD9B deficiency dysregulates cell adhesion genes during neural organoid formation . Use RAD9B antibodies alongside antibodies for these adhesion proteins to validate changes at the protein level.

• Temporal Correlation Analysis:

  • Perform time-course experiments collecting both RNA and protein samples

  • Correlate changes in RAD9B mRNA expression with protein levels to identify potential post-transcriptional regulation

  • Monitor downstream targets identified through RNA-seq (particularly PAX6 and OCT4) at both RNA and protein levels

• Localization-Expression Relationship:

  • Use subcellular fractionation followed by immunoblotting to determine if changes in localization precede or follow expression changes

  • Compare wild-type vs. variant RAD9B nuclear localization and correlate with expression of differentially regulated genes

• Causal Relationship Testing:

  • After identifying dysregulated pathways through RNA-seq, use RAD9B antibodies in ChIP assays to determine if RAD9B directly regulates these genes

  • Use immunoprecipitation to identify protein complexes that might mediate between RAD9B and the dysregulated pathways

• Model System Validation:

  • Compare results between 2D cell cultures and 3D organoids

  • Use immunohistochemistry with RAD9B antibodies to localize expression in developing neural structures

This integrated approach can reveal whether changes in gene expression are directly or indirectly related to RAD9B function, providing deeper insights into its developmental role.

What emerging applications of RAD9B antibodies might advance understanding of cancer biology?

Emerging applications of RAD9B antibodies in cancer research include:

• Tumor Suppressor Screening: Since reduced HRAD9B expression has been observed in testicular tumors, particularly seminomas , RAD9B antibodies could be used in tissue microarrays to screen various cancer types for altered expression.

• Genomic Instability Biomarker Development: As RAD9B is linked to genomic stability , antibody-based assays could help identify cancers with DNA repair defects amenable to specific therapeutic approaches.

• Therapy Response Prediction:

  • Develop immunohistochemistry panels including RAD9B to predict response to DNA-damaging chemotherapeutics

  • Monitor RAD9B expression and localization changes during treatment to understand adaptation mechanisms

• Combined DNA Damage Response Profiling:

  • Create multiplexed antibody panels targeting RAD9B alongside other DDR proteins

  • Correlate RAD9B status with mutation signatures to identify potential synthetic lethal relationships

• Circulating Tumor Cell Analysis: Develop sensitive detection methods using RAD9B antibodies to identify CTCs with specific DNA repair deficiencies.

This research direction aligns with the understanding that RAD9B dysregulation has been linked to genomic instability and cancer development , potentially opening new avenues for targeted cancer therapies.

How might neural organoid studies benefit from advanced RAD9B antibody applications?

Advanced RAD9B antibody applications can enhance neural organoid studies through:

• Developmental Trajectory Mapping: Use RAD9B antibodies in time-course immunohistochemistry to map expression changes during critical developmental windows of neural organoid formation, especially since RAD9B deficiency impedes in vitro formation of neural organoids .

• Multi-parameter Imaging:

  • Combine RAD9B antibodies with markers for neural progenitors, mature neurons, and cell adhesion molecules

  • Apply clearing techniques and 3D imaging to visualize RAD9B distribution throughout intact organoids

• Single-Cell Protein Analysis:

  • Use RAD9B antibodies in mass cytometry or imaging mass cytometry to correlate RAD9B levels with cell state in heterogeneous organoid populations

  • Identify specific cell populations where RAD9B function is most critical

• Functional Genomics Integration:

  • Apply RAD9B antibodies in ChIP-seq experiments using organoid material to identify direct genomic targets

  • Correlate with RNA-seq data showing dysregulation of cell adhesion genes during organoid formation

• Disease Modeling:

  • Apply RAD9B antibodies to compare expression and localization in organoids derived from neural tube defect patients versus controls

  • Investigate whether RAD9B variants associated with spina bifida (8/409 cases vs. 0/298 controls) affect protein function in patient-derived organoids

These approaches would contribute to understanding how RAD9B influences neural development and potentially identify therapeutic targets for neurodevelopmental disorders associated with RAD9B dysfunction.

What methodological advances might improve detection of low-abundance RAD9B in non-testicular tissues?

To enhance detection of low-abundance RAD9B in tissues beyond its predominant testicular expression :

• Signal Amplification Technologies:

  • Implement tyramide signal amplification (TSA) with RAD9B antibodies to enhance sensitivity in immunohistochemistry/immunofluorescence

  • Use proximity ligation assays (PLA) to detect RAD9B interactions with known binding partners, amplifying detection signal

• Sample Enrichment Techniques:

  • Apply subcellular fractionation to concentrate nuclear RAD9B before immunoblotting

  • Use immunoprecipitation to concentrate RAD9B from large tissue samples before detection

• Highly-Sensitive Protein Detection Methods:

  • Apply digital ELISA (Simoa) technology for single-molecule RAD9B detection

  • Implement mass spectrometry-based targeted proteomics using RAD9B antibodies for immunocapture

• Contextual Expression Enhancement:

  • Induce DNA damage in experimental systems to potentially upregulate RAD9B expression

  • Examine tissues during developmental windows when RAD9B may be transiently expressed at higher levels, particularly in neural tissues

• Alternative Antibody Formats:

  • Develop nanobodies or single-chain antibody fragments with potentially enhanced epitope access

  • Create bispecific antibodies targeting RAD9B and common binding partners to improve avidity

These methodological advances would particularly benefit research examining RAD9B's role in neural development and DNA damage response in diverse tissue contexts.

What is known about the functional impact of specific RAD9B variants detected in neural tube defect cases?

Research has identified several RAD9B variants in spina bifida cases with distinct functional impacts:

Methodological considerations when studying these variants:

• Nuclear Localization Assessment: Use subcellular fractionation followed by Western blotting with RAD9B antibodies or immunofluorescence microscopy to determine variant protein distribution.

• Protein Stability Analysis: Perform cycloheximide chase experiments with RAD9B antibody detection to assess variant protein half-life.

• Proliferation Studies: Monitor cell growth rates in systems expressing wild-type versus variant RAD9B.

• Signaling Pathway Analysis: Use phospho-specific antibodies to detect JNK phosphorylation levels in conjunction with RAD9B expression.

• DNA Damage Response: Assess formation of DNA damage foci under oxidative stress conditions using immunofluorescence with RAD9B antibodies.