RAD9B Antibody, HRP conjugated

What is RAD9B and why are antibodies against it important in research?

RAD9B (RAD9 checkpoint clamp component B) is a protein involved in DNA repair and DNA replication processes. The canonical human RAD9B protein has 426 amino acid residues with a molecular mass of approximately 47.8 kDa . As a member of the Rad9 protein family, RAD9B is part of the 9-1-1 (RAD9-RAD1-HUS1) heterotrimeric complex that forms a checkpoint clamp with structural resemblance to PCNA (proliferating cell nuclear antigen) .

RAD9B antibodies are critical research tools because they enable:

• Detection and localization of RAD9B in various tissues and cellular compartments

• Monitoring RAD9B expression levels in different experimental conditions

• Investigating RAD9B's role in DNA damage response pathways

• Studying protein-protein interactions involving RAD9B

• Examining potential alterations in checkpoint regulation in disease models

RAD9B is primarily expressed in testis and skeletal muscle, making these antibodies particularly valuable for reproductive biology and muscle physiology research .

What is the difference between unconjugated RAD9B antibodies and HRP-conjugated versions?

Unconjugated RAD9B antibodies and HRP-conjugated versions differ in several important aspects:

The HRP conjugation provides direct enzymatic activity for signal generation in assays that utilize peroxidase substrates, eliminating the need for a secondary antibody step. This makes HRP-conjugated RAD9B antibodies particularly valuable for streamlined protocols in Western blotting and immunohistochemistry applications .

What experimental applications are most suitable for RAD9B antibody, HRP conjugated?

HRP-conjugated RAD9B antibodies are particularly well-suited for several key experimental applications:

• Western Blotting: HRP-conjugated RAD9B antibodies enable direct detection of RAD9B protein after gel electrophoresis and membrane transfer, eliminating the need for secondary antibody incubation. This application is especially valuable when monitoring RAD9B expression levels across different experimental conditions or tissue types .

• Immunohistochemistry (IHC): For fixed tissue sections, HRP-conjugated RAD9B antibodies allow direct visualization of RAD9B protein localization through chromogenic substrates like DAB (3,3'-diaminobenzidine), particularly useful for studying RAD9B expression patterns in testis and skeletal muscle tissues .

• ELISA (Enzyme-Linked Immunosorbent Assay): The direct HRP conjugation facilitates quantitative measurement of RAD9B levels in various sample types with reduced protocol complexity and background signal .

• Flow Cytometry: When studying intracellular RAD9B expression in cell populations, HRP-conjugated antibodies can be used with appropriate permeabilization and substrate systems.

• Chromatin Immunoprecipitation (ChIP) Assays: For studying RAD9B interactions with DNA and chromatin-associated proteins, especially in checkpoint activation contexts .

When selecting appropriate experimental applications, researchers should consider that the molecular weight of human RAD9B (47.8 kDa) may vary due to the presence of multiple isoforms (up to 5 reported variants) and potential post-translational modifications .

How can RAD9B antibody, HRP conjugated be used to investigate the 9-1-1 checkpoint clamp complex interactions?

HRP-conjugated RAD9B antibodies can be strategically employed to investigate the complex interactions within the 9-1-1 checkpoint clamp through several advanced methodologies:

• Co-immunoprecipitation with Direct Detection: Using HRP-conjugated RAD9B antibodies in co-IP experiments allows researchers to pull down RAD9B and its interacting partners (RAD1, HUS1), followed by direct detection of RAD9B in the complex. This approach can be particularly valuable when studying how the hydrophobic pocket on the front side of RAD9 participates in intramolecular interactions with the RAD9 C-tail .

• Proximity Ligation Assays (PLA): HRP-conjugated RAD9B antibodies can be paired with antibodies against potential interacting proteins like RHINO, which binds to the hydrophobic pocket of RAD9. The signal generated from the HRP can indicate proximity between RAD9B and its binding partners in fixed cells or tissues .

• Chromatin Fractionation Studies: By using HRP-conjugated RAD9B antibodies for direct detection in chromatin fractionation experiments, researchers can monitor the recruitment of RAD9B to DNA damage sites and its association with chromatin during checkpoint activation.

• Domain-Specific Interaction Mapping: When paired with deletion mutant analysis, HRP-conjugated RAD9B antibodies can help map which domains of RAD9B (particularly the PCNA-like domain) are involved in specific protein-protein interactions within the quaternary complex of 9-1-1-RHINO .

• Time-Course Studies of Complex Formation: The direct detection capabilities of HRP-conjugated antibodies allow for efficient kinetic studies of how the 9-1-1 complex assembly changes over time following DNA damage.

These approaches leverage the structural insights that RAD9 contains a distinctive C-terminal intrinsically disordered region (C-tail) that distinguishes it from other components of the heterotrimeric ring structure, making it a key regulatory component of checkpoint activation .

What methodological challenges exist when using RAD9B antibody, HRP conjugated for detecting different RAD9B isoforms?

Detecting different RAD9B isoforms with HRP-conjugated antibodies presents several methodological challenges that researchers must address through careful experimental design:

• Epitope Accessibility Across Isoforms: With up to five different isoforms reported for RAD9B, HRP-conjugated antibodies may have variable access to their target epitopes depending on protein folding differences between isoforms. Researchers should carefully select antibodies targeting epitopes conserved across isoforms of interest .

• Molecular Weight Resolution: Distinguishing between isoforms that may have similar molecular weights requires:

  • Using high-resolution SDS-PAGE gels (10-12%) with extended running times

  • Employing gradient gels (4-20%) to maximize separation of closely migrating isoforms

  • Optimizing transfer conditions for proteins across the entire relevant molecular weight range

• Cross-Reactivity Management: HRP-conjugated RAD9B antibodies may cross-react with the related RAD9A protein or other family members. Control experiments comparing wild-type and knockdown/knockout samples are essential for validation.

• Isoform-Specific Detection Strategies: For targeted isoform analysis, researchers can implement:

• Signal Quantification Challenges: HRP signal development must be carefully optimized to remain in the linear range for accurate quantification of potentially low-abundance isoforms, particularly when analyzing multiple isoforms simultaneously.

When addressing these challenges, researchers should note that RAD9B gene orthologs have been identified in multiple species (mouse, rat, bovine, frog, chimpanzee, and chicken), which can provide comparative models for isoform-specific studies .

How can the structural insights about RAD9's hydrophobic pocket guide experimental design when using RAD9B antibody, HRP conjugated?

Recent structural analyses have revealed that the RAD9 protein contains a significant hydrophobic pocket involved in both intramolecular interactions with its own C-tail and intermolecular interactions with partners like RHINO . This knowledge can strategically guide experimental design when using HRP-conjugated RAD9B antibodies:

• Epitope-Aware Antibody Selection: Researchers should consider selecting HRP-conjugated RAD9B antibodies with epitopes that either:

  • Target regions outside the hydrophobic pocket to avoid interference with natural interactions

  • Specifically target the hydrophobic pocket to competitively inhibit interactions

  • Recognize the C-tail region to monitor its availability for interactions

• Domain-Specific Interaction Studies: The identified hydrophobic pocket creates opportunities for domain-focused experiments:

• Checkpoint Activation Monitoring: Design experiments that use HRP-conjugated RAD9B antibodies to track how the availability of the hydrophobic pocket changes during DNA damage response:

  • Time-course studies following DNA damage induction

  • Chromatin fractionation to monitor pocket-dependent recruitment

  • Co-localization studies with known interacting partners

• Structure-Based Inhibitor Screening: Leverage the structural insights to develop screening assays where:

  • HRP-conjugated RAD9B antibodies detect displacement of natural binding partners

  • Signal changes indicate potential therapeutic compound binding to the pocket

• Conformational Change Analysis: Monitor potential conformational changes in RAD9B structure by:

  • Comparing HRP-antibody accessibility in native versus denatured conditions

  • Using limited proteolysis followed by HRP-antibody detection to identify protected regions

By integrating these structural insights into experimental design, researchers can develop more targeted approaches to studying RAD9B's role in the 9-1-1-RHINO quaternary complex and its functions in checkpoint activation .

What are the optimal conditions for using RAD9B antibody, HRP conjugated in Western blot applications?

Optimizing Western blot protocols for HRP-conjugated RAD9B antibodies requires attention to several key parameters:

• Sample Preparation:

  • Use RIPA buffer supplemented with protease inhibitors for efficient extraction of RAD9B

  • Include phosphatase inhibitors to preserve phosphorylation states of RAD9B

  • Optimal protein loading: 20-40 μg of total protein from cell lysates, 50-80 μg from tissue extracts

• Gel Selection and Transfer Conditions:

• Antibody Incubation Protocol:

  • Optimal dilution: 1:1000 to 1:2000 (adjust based on specific antibody concentration)

  • Incubation time: 2 hours at room temperature or overnight at 4°C

  • Washing: 5 × 5 minutes with TBST containing 0.1% Tween-20

  • Positive control: Testis or skeletal muscle lysates (known to express RAD9B)

• Signal Development Optimization:

  • For chemiluminescence: Incubate membrane with substrate for 1 minute exactly

  • For multiple exposures: 30 seconds, 1 minute, and 3 minutes

  • Use film or digital imaging systems with wide dynamic range capability

  • Perform signal linearity validation with concentration curve of positive control

• Troubleshooting Common Issues:

  • Multiple bands: May indicate isoforms (validate with isoform-specific controls)

  • Weak signal: Increase antibody concentration, extend incubation time, or use signal enhancers

  • High background: Increase washing stringency, optimize blocking conditions

  • Non-specific bands: Validate with knockout/knockdown controls or peptide competition

When analyzing results, researchers should be aware that RAD9B has up to 5 different reported isoforms that may appear as distinct bands on the blot, with the canonical form at approximately 47.8 kDa .

How should researchers validate the specificity of RAD9B antibody, HRP conjugated before experimental use?

Thorough validation of HRP-conjugated RAD9B antibodies is critical for ensuring reliable experimental results. A comprehensive validation strategy includes:

• Positive and Negative Control Samples:

  • Positive controls: Testis and skeletal muscle extracts (known to express RAD9B)

  • Negative controls: Tissues with minimal RAD9B expression or RAD9B knockout/knockdown samples

  • Recombinant protein control: Purified recombinant RAD9B for absolute specificity benchmarking

• Peptide Competition Assay:

  • Pre-incubate HRP-conjugated RAD9B antibody with excess synthetic peptide corresponding to the target epitope

  • Run parallel experiments with blocked and unblocked antibody

  • Specific signals should be significantly reduced or eliminated in the peptide-blocked condition

• Cross-Reactivity Assessment:

• Immunodepletion Experiments:

  • Serially deplete RAD9B from samples using the antibody

  • Analyze both depleted sample and immunoprecipitate

  • HRP-conjugated antibody should show diminishing signal in depleted samples

• Orthogonal Method Validation:

  • Compare results using HRP-conjugated RAD9B antibody with other detection methods:

    • Mass spectrometry identification of detected bands

    • RNA expression correlation (qPCR for RAD9B transcript)

    • Immunofluorescence co-localization with differently-targeted RAD9B antibodies

• Functional Validation:

  • Perform immunoprecipitation followed by activity assays to confirm that the antibody captures functionally active RAD9B

  • Verify that precipitated complexes maintain known interactions with RAD1 and HUS1

By implementing this comprehensive validation strategy, researchers can ensure that their HRP-conjugated RAD9B antibody specifically detects the intended target and provides reliable results in their experimental systems .

What protocols should be followed when using RAD9B antibody, HRP conjugated for studying DNA damage response pathways?

When investigating DNA damage response pathways using HRP-conjugated RAD9B antibodies, researchers should implement the following optimized protocols:

• DNA Damage Induction and Time-Course Analysis:

• Subcellular Fractionation Protocol:

  • Separate nuclear, chromatin-bound, and cytoplasmic fractions using detergent-based extraction

  • Analyze RAD9B distribution using HRP-conjugated antibody at 1:1000 dilution

  • Include markers for each fraction (GAPDH for cytoplasmic, Histone H3 for chromatin)

  • Quantify relative distribution changes following DNA damage induction

• 9-1-1 Complex Analysis Protocol:

  • Perform reciprocal co-immunoprecipitation using antibodies against RAD1 or HUS1

  • Detect RAD9B using HRP-conjugated antibody at 1:2000 dilution

  • Analyze complex formation kinetics following DNA damage

  • Include phosphatase treatment controls to assess phosphorylation-dependent interactions

• RHINO Interaction Study Protocol:

  • Design proximity ligation assays using HRP-RAD9B antibody and anti-RHINO antibody

  • Analyze co-localization at DNA damage sites using confocal microscopy

  • Quantify interaction signals at different time points after damage

  • Compare wild-type interactions with hydrophobic pocket mutants

• Chromatin Immunoprecipitation (ChIP) Protocol for RAD9B:

  • Crosslink proteins to DNA using 1% formaldehyde for 10 minutes

  • Sonicate chromatin to 200-500 bp fragments

  • Immunoprecipitate with HRP-conjugated RAD9B antibody (use anti-HRP beads for pull-down)

  • Analyze RAD9B recruitment to specific genomic loci by qPCR

  • Focus on origins of replication and common fragile sites

• Fluorescence Recovery After Photobleaching (FRAP) Analysis:

  • Transfect cells with fluorescently-tagged RAD9B

  • Validate expression pattern using HRP-conjugated RAD9B antibody

  • Perform FRAP analysis before and after DNA damage

  • Quantify changes in RAD9B mobility at damage sites

These protocols should be adapted based on the specific cell types being studied, with particular attention to tissues known to express RAD9B (testis and skeletal muscle) . Researchers should also consider the presence of multiple isoforms and potential differences in their recruitment to damage sites .

How can researchers troubleshoot inconsistent results when using RAD9B antibody, HRP conjugated?

When encountering inconsistent results with HRP-conjugated RAD9B antibodies, researchers should systematically address potential issues:

• Antibody-Related Variables:

• Sample Preparation Troubleshooting:

  • Protein degradation: Add fresh protease inhibitors; maintain samples at 4°C; reduce processing time

  • Incomplete extraction: Try alternate lysis buffers (RIPA, NP-40, Triton X-100) to optimize RAD9B extraction

  • Post-translational modifications: Add phosphatase inhibitors; compare treatments affecting modification state

  • Expression variability: Standardize culture conditions; control cell confluence and passage number

• Technical Protocol Optimization:

  • Signal development: Standardize exposure times; use automated systems for consistency

  • Buffer composition: Test multiple blocking agents (milk, BSA, commercial blockers)

  • Temperature control: Maintain consistent temperature during all incubations

  • Wash stringency: Optimize wash buffer composition and duration

• Biological Variable Management:

  • Cell cycle dependence: Synchronize cells; analyze results with cell cycle markers

  • Stress response effects: Control environmental variables; document cell stress indicators

  • Isoform-specific detection: Use controls expressing specific isoforms; document which isoforms are targeted

• Systematic Validation Strategy:

  • Use orthogonal detection methods to confirm RAD9B behavior

  • Implement genetic controls (siRNA, CRISPR knockout) for specificity validation

  • Perform parallel analyses with multiple antibodies targeting different RAD9B epitopes

  • Document experimental conditions in detail for reproducibility

When working with DNA damage response experiments specifically, researchers should implement rigorous controls for damage induction, as variability in damage levels can dramatically affect RAD9B recruitment and complex formation with RAD1 and HUS1 .

How can researchers use RAD9B antibody, HRP conjugated for multiplex detection systems with other checkpoint proteins?

Multiplex detection systems incorporating HRP-conjugated RAD9B antibodies with other checkpoint proteins require sophisticated experimental design:

• Sequential Multiplex Western Blotting:

• Multiplex Immunofluorescence Strategies:

  • Tyramide Signal Amplification (TSA): Use HRP-conjugated RAD9B antibody to catalyze deposition of fluorescent tyramide; heat-inactivate HRP before subsequent rounds

  • Spectral unmixing: Combine HRP-substrates with different fluorescent emissions for simultaneous detection

  • Sequential microwave treatment: Allows reuse of the same secondary antibody system with different primaries

• Proximity-Based Multiplex Systems:

  • Proximity Ligation Assay (PLA): Combine HRP-RAD9B antibody with antibodies against RAD1, HUS1, or RHINO

  • CODEX system: Use HRP-conjugated RAD9B antibody with DNA-barcoded antibodies against other checkpoint proteins

  • 4i (iterative indirect immunofluorescence imaging): Combine with cyclic immunofluorescence for high-dimensional analysis

• Quantitative Multiplex Application Design:

• Controls for Multiplex Systems:

  • Single-antibody controls to establish baseline signals

  • Blocking peptide controls to verify specificity

  • Order-of-addition controls to exclude artifacts

  • Channel bleed-through controls for fluorescence applications

By implementing these multiplex strategies, researchers can simultaneously analyze RAD9B in context with other components of the 9-1-1 checkpoint clamp (RAD1, HUS1) and interacting proteins like RHINO, providing comprehensive insights into checkpoint complex dynamics during DNA damage responses .

What are the emerging applications of RAD9B antibody, HRP conjugated in single-cell analysis of DNA damage responses?

Single-cell analysis represents a frontier in DNA damage response research, with HRP-conjugated RAD9B antibodies enabling several innovative applications:

• Imaging Mass Cytometry Applications:

  • Metal-tagged RAD9B antibodies can be used alongside dozens of other checkpoint markers

  • Spatial distribution of RAD9B at damage sites can be correlated with cell cycle markers

  • High-dimensional analysis can reveal previously unrecognized cell subpopulations with distinct RAD9B responses

• Single-Cell Western Blot Adaptations:

• Flow Cytometry-Based Single-Cell Applications:

  • Use HRP-conjugated RAD9B antibody with fluorescent substrates for flow cytometry

  • Combine with DNA damage markers (γH2AX) and cell cycle indicators

  • Implement index sorting for downstream single-cell genomics/transcriptomics

  • Design branched DNA signal amplification for rare isoform detection

• Single-Cell Genomics Integration:

  • CITE-seq adaptation using HRP-conjugated RAD9B antibody with oligonucleotide tags

  • Correlation of RAD9B protein levels with single-cell transcriptomes

  • RAD9B complex immunoprecipitation from single cells followed by DNA sequencing

  • Chromatin accessibility correlation with RAD9B binding at single-cell resolution

• Advanced Microscopy Applications:

  • Use HRP-mediated proximity labeling to identify RAD9B-proximal proteins in single cells

  • Implement live-cell sensors to correlate with fixed-cell HRP-RAD9B antibody staining

  • Apply optical tweezers alongside HRP-RAD9B detection to study mechanical aspects of damage response

  • Integrate with DNA damage site labeling systems for spatiotemporal resolution

These emerging applications can provide insights into cell-to-cell variability in checkpoint activation, revealing how individual cells within a population may respond differently to DNA damage. This heterogeneity could have significant implications for understanding treatment responses in contexts like cancer therapy, where DNA damage-inducing agents are commonly employed.

The integration of HRP-conjugated RAD9B antibody detection with single-cell multi-omics approaches represents a particularly promising frontier, as it allows for correlation between protein-level checkpoint activities and underlying genomic or epigenomic features that may influence damage response pathways .

What are the key considerations for researchers selecting RAD9B antibody, HRP conjugated for their research?

When selecting HRP-conjugated RAD9B antibodies for research applications, investigators should evaluate several critical factors to ensure optimal experimental outcomes:

• Specificity Considerations:

  • Epitope location relative to functional domains (PCNA-like domain vs. C-tail)

  • Cross-reactivity profile against RAD9A and other related proteins

  • Isoform specificity (considering the 5 reported RAD9B isoforms)

  • Species reactivity for comparative studies (human, mouse, rat, etc.)

• Technical Performance Metrics:

• Application-Specific Selection Criteria:

  • For western blotting: Select antibodies validated specifically for denatured epitopes

  • For immunoprecipitation: Choose antibodies recognizing native conformations

  • For IHC applications: Prioritize antibodies validated for specific fixation methods

  • For multiplex applications: Consider antibodies with demonstrated compatibility in multiplex systems

• Experimental Design Alignment:

  • Align epitope selection with experimental questions about RAD9B function

  • Consider whether the hydrophobic pocket region should be targeted or avoided

  • Select conjugation ratio appropriate for the expected abundance of RAD9B

  • Evaluate whether the antibody can detect post-translationally modified forms

• Validation Documentation Requirements:

  • Review published validation data from manufacturers

  • Assess independent validation in peer-reviewed literature

  • Request validation data in cell/tissue types relevant to planned experiments

  • Consider antibodies validated by orthogonal methods (mass spectrometry, genetic knockout)

By systematically evaluating these factors, researchers can select HRP-conjugated RAD9B antibodies that will provide reliable and reproducible results in their specific experimental systems, enabling robust investigations of RAD9B's roles in DNA damage checkpoint regulation .

How is our understanding of RAD9B's role in checkpoint regulation evolving, and what implications does this have for antibody-based research?

Our understanding of RAD9B's role in checkpoint regulation is rapidly evolving, creating new opportunities and challenges for antibody-based research:

• Emerging Structural Insights:

  • Recent structural studies have revealed the importance of RAD9's hydrophobic pocket in both intra- and intermolecular interactions

  • The RAD9 C-tail has been shown to interact with its own PCNA-like domain, suggesting autoregulatory mechanisms

  • RHINO binding to the hydrophobic pocket of RAD9 provides new insights into checkpoint complex assembly

  • These structural details enable more targeted antibody selection and experimental design

• Functional Complexity Beyond Classical Checkpoints:

• Isoform-Specific Functions:

  • Growing evidence suggests that the five reported RAD9B isoforms may have distinct functions

  • Antibody-based research must evolve to distinguish between these isoforms

  • Development of isoform-specific antibodies will be crucial for understanding specialized roles

  • Integration of antibody detection with isoform-specific genetic approaches offers powerful opportunities

• Integration with Emerging Technologies:

  • CRISPR-based tagging strategies can complement antibody approaches

  • Proximity labeling methods using HRP-conjugated antibodies enable discovery of novel interactions

  • Single-molecule imaging approaches reveal dynamic behavior not accessible to traditional antibody methods

  • Multi-omics integration provides systems-level context for RAD9B functions

• Therapeutic Relevance and Translational Applications:

  • RAD9B checkpoint functions may represent therapeutic targets in cancer contexts

  • Antibody-based screening of small molecule inhibitors targeting RAD9B interactions

  • Potential prognostic value of RAD9B expression/localization patterns in cancer samples

  • Development of RAD9B function-blocking antibodies as potential therapeutic tools

These evolving insights highlight that RAD9B is not merely a structural component of the 9-1-1 checkpoint clamp but a dynamic regulator with multiple interaction surfaces and potential functions. The hydrophobic pocket on RAD9's front side appears to be a particularly important regulatory site, mediating both intramolecular interactions with its own C-tail and intermolecular interactions with proteins like RHINO .