RAD23B Monoclonal Antibody

What is RAD23B and what are its known functions?

RAD23B, also known as p58, hHR23B, or HR23B, belongs to the RAD23 family and serves as a critical component in multiple cellular pathways. It plays a central role in both proteasomal degradation of misfolded proteins and DNA repair mechanisms . As a key component of nucleotide excision repair (NER), RAD23B interacts with the NER factor Rad4, and this dimer can bind damaged DNA . Additionally, RAD23B serves as a central component of a complex required to couple deglycosylation and proteasome-mediated degradation of misfolded proteins in the endoplasmic reticulum that undergo retrotranslocation .

What applications are RAD23B monoclonal antibodies validated for?

RAD23B monoclonal antibodies have been validated for multiple research applications:

These applications allow researchers to examine RAD23B expression, localization, and interactions in various experimental contexts .

What is the molecular weight of RAD23B and why might observed values differ?

Although the calculated molecular weight of RAD23B is 43 kDa (409 amino acids), the observed molecular weight in SDS-PAGE and Western blot applications is consistently reported as 58 kDa . This discrepancy between calculated and observed molecular weights is common for many proteins and may result from post-translational modifications, structural features that affect migration in gels, or the presence of unprocessed regions. When validating a new RAD23B antibody, researchers should expect to observe a band at approximately 58 kDa rather than at the calculated 43 kDa position .

What species reactivity is reported for commercial RAD23B monoclonal antibodies?

Commercial RAD23B monoclonal antibodies demonstrate cross-reactivity with multiple species:

This cross-reactivity information is crucial when designing experiments using tissue or cells from different species. Researchers should verify the specific reactivity pattern of their selected antibody before proceeding with experiments .

How does RAD23B's dual role in protein degradation and DNA repair manifest in experimental systems?

RAD23B's dual functionality creates unique experimental challenges and opportunities. Studies have demonstrated that RAD23B participates in two distinct NER events. The interaction between RAD23B and Rad4 is critical for stabilizing Rad4, and a small amino acid domain (the R4B domain) is sufficient for this interaction . This domain alone can stabilize Rad4 and promote NER.

Additionally, genetic studies indicate that RAD23B functions in a separate but concurrent activity in NER that involves interaction with the proteasome. This proteasome-related activity is distinct from its role in stabilizing Rad4 . Remarkably, simultaneous expression of both mutant forms of the RAD23B protein can restore full resistance to UV light, suggesting complementary functions.

When designing experiments to study either function, researchers should consider:

• Using domain-specific antibodies to discriminate between protein degradation and DNA repair functions

• Employing cellular stress that specifically activates one pathway (e.g., proteasome inhibitors versus UV damage)

• Creating function-specific knockouts rather than complete RAD23B deletion

What are the optimal antigen retrieval methods for RAD23B immunohistochemistry?

For optimal antigen retrieval in RAD23B immunohistochemistry applications, the following protocols have been validated:

The choice between these retrieval methods may depend on tissue type, fixation protocol, and specific antibody used. For mouse brain tissue, TE buffer at pH 9.0 is generally recommended as the primary approach, with citrate buffer at pH 6.0 serving as an alternative when necessary . For human breast cancer tissue, citrate buffer at pH 6.0 has been successfully employed . Researchers should optimize the antigen retrieval protocol for their specific tissue type and fixation conditions.

How can I optimize my immunoblotting procedure for RAD23B detection in different sample types?

Optimizing immunoblotting for RAD23B requires attention to several technical parameters that vary by sample type:

• Sample preparation: For cellular samples (e.g., HeLa, HEK-293), standard RIPA buffer with protease inhibitors is effective. For tissue samples (e.g., brain tissue), additional mechanical homogenization may be necessary .

• Dilution optimization:

  • For cellular samples: Start with 1:5000 dilution for monoclonal antibodies

  • For tissue samples: More concentrated antibody may be required (1:1000-1:3000)

  • For polyclonal antibodies: Generally use 1:500-1:3000 range

• Detection considerations:

  • Secondary antibody selection: A 1:2000 dilution of goat anti-mouse horseradish peroxidase conjugate has been successfully used

  • Development method: Enhanced chemiluminescence reagents provide good sensitivity

  • Expected band: Look for the 58 kDa band, not the calculated 43 kDa size

• Buffer system: PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 has been reported as effective for antibody stability

Careful optimization of these parameters will help ensure specific detection of RAD23B across different experimental systems.

What controls should be included when performing immunoprecipitation with RAD23B antibodies?

When designing immunoprecipitation experiments with RAD23B antibodies, the following controls are essential:

• Input control: Reserve 5-10% of the pre-immunoprecipitation lysate to verify protein expression and loading .

• Negative controls:

  • IgG control: Use the same isotype (IgG2a for mouse monoclonal antibodies) at equivalent concentration

  • Non-expressing cells/tissues: Include samples known to have low or no RAD23B expression

  • Immunoprecipitation with pre-immune serum for polyclonal antibodies

• Positive controls:

  • Tagged RAD23B constructs: Consider using myc-tagged or FLAG-tagged RAD23B constructs in parallel experiments

  • Known RAD23B interactors: Check for co-immunoprecipitation of known binding partners like Rad4

• Validation of specificity:

  • Perform reciprocal immunoprecipitations where possible

  • Verify results with different antibody clones if available

For detection of immunoprecipitated complexes, follow established protocols using anti-FLAG, anti-myc, or specific antibodies for interacting proteins, and employ a 1:2000 dilution of goat anti-mouse horseradish peroxidase conjugate with enhanced chemiluminescence reagents for visualization .

How should I address the molecular weight discrepancy between calculated and observed RAD23B in my results?

The discrepancy between RAD23B's calculated molecular weight (43 kDa) and observed molecular weight (58 kDa) requires careful consideration in experimental design and data interpretation :

• Experimental verification approaches:

  • Run positive control samples alongside experimental samples

  • Use multiple antibodies targeting different epitopes to confirm the 58 kDa band's identity

  • Consider using tagged recombinant RAD23B as an additional size reference

  • If available, include samples from RAD23B knockout models as negative controls

• Technical explanations to consider:

  • Post-translational modifications: RAD23B undergoes modifications that affect migration

  • Structural elements: Certain domains may resist complete denaturation

  • Splice variants: Alternative splicing may result in larger protein species

  • Experimental conditions: SDS-PAGE parameters (buffer composition, gel percentage) can affect migration

• Documentation requirements:

  • Always report both calculated and observed molecular weights in publications

  • Specify the antibody clone, dilution, and detection method used

  • Include complete Western blot images with molecular weight markers visible

Understanding this consistent size discrepancy is important for accurate data interpretation and experimental troubleshooting .

What protocols are recommended for multiplexed detection of RAD23B and its interaction partners?

For investigating RAD23B interactions through multiplexed detection approaches:

• Co-immunoprecipitation followed by immunoblotting:

  • Prepare cellular extracts using binding buffer (composition should be optimized for specific interaction)

  • Perform immunoprecipitation with beads linked to specific antibodies

  • Use SDS-8% PAGE for separation

  • Conduct sequential immunoblotting with anti-FLAG, anti-myc, or other epitope tag antibodies

  • For proteasome interactions, probe with anti-Rpt5 antibodies in addition to RAD23B-specific antibodies

• Immunofluorescence co-localization:

  • Fix HeLa or other suitable cells using standard protocols

  • Utilize RAD23B antibody (e.g., clone SAIC-28A-29) at optimized dilution

  • Counter-stain with DAPI for nuclear visualization

  • Include antibodies against interaction partners of interest

  • Use confocal microscopy for co-localization analysis

• Flow cytometry approach:

  • Prepare single-cell suspensions from appropriate cell lines

  • Use validated RAD23B antibodies (e.g., clone SAIC-28A-29)

  • Consider multiplexed staining with additional proteins of interest

  • Analyze using standard flow cytometry methods

These approaches allow investigation of RAD23B's diverse interactions in both the DNA repair and protein degradation pathways.

Troubleshooting and Data Interpretation

Validating RAD23B specificity in immunofluorescence requires multiple approaches:

• Positive and negative controls:

  • Include HeLa cells as positive controls (verified to express RAD23B)

  • Use siRNA or shRNA knockdown of RAD23B as negative controls

  • Consider cell lines with known differential expression of RAD23B

• Antibody validation strategies:

  • Compare staining patterns across multiple RAD23B antibody clones

  • Test dilution series (1:400 to 1:1600) to identify optimal signal-to-noise ratio

  • Perform absorption controls with immunizing peptide when available

• Co-localization approaches:

  • Check for expected subcellular localization patterns

  • Co-stain with markers for structures where RAD23B functions (e.g., proteasome markers)

  • Use DAPI counterstaining to evaluate nuclear localization

• Technical optimization:

  • Test multiple fixation methods (paraformaldehyde, methanol)

  • Optimize permeabilization conditions

  • Include appropriate blocking reagents to minimize non-specific binding

Successful immunofluorescence applications with RAD23B antibodies have been reported in HeLa cells, providing a reliable positive control system for protocol optimization .

What factors affect RAD23B protein levels in experimental systems?

Multiple factors can influence RAD23B protein levels, which researchers should consider when designing experiments:

• Cell cycle and stress conditions:

  • DNA damage (particularly UV exposure) may alter RAD23B expression or localization

  • Proteasome inhibitors can affect RAD23B stability and function in protein degradation pathways

  • Cell cycle phase may influence expression levels

• Genetic factors:

  • Loss of ubiquitin-conjugating enzymes (Ubc4/Ubc5) affects RAD23B's role in stabilizing Rad4

  • Mutations in interaction domains can alter protein stability without affecting expression

  • Splicing variations may produce different isoforms with altered stability

• Experimental variables:

  • Extraction methods influence recovery (e.g., RIPA buffer vs. specialized extraction protocols)

  • Sample handling (freeze-thaw cycles, protease inhibitor inclusion) affects degradation

  • Culture conditions for cell lines (confluency, passage number) may alter expression

• Tissue-specific considerations:

  • Expression varies across tissues (brain tissue shows consistent expression)

  • Cancer cells may exhibit altered expression patterns compared to normal tissues

  • Species differences exist but cross-reactivity is observed across human, mouse, rat, and pig samples

Understanding these variables is essential for proper experimental design and interpretation of RAD23B detection results.

How can I investigate the differential roles of RAD23B in DNA repair versus protein degradation pathways?

To distinguish between RAD23B's dual functions, consider these experimental approaches:

• Domain-specific disruption:

  • Target the Rad4-binding (R4B) domain to specifically affect DNA repair function

  • Disrupt UBA domains to preferentially impact protein degradation pathways

  • Express individual domains as competitors to distinguish functions

• Pathway-specific assays:

  • UV sensitivity assays primarily reflect DNA repair function

  • Proteotoxic stress response assays highlight protein degradation role

  • Measure nucleotide excision repair capacity versus proteasomal degradation separately

• Interaction-specific analysis:

  • Immunoprecipitation followed by mass spectrometry to identify context-specific binding partners

  • Use differential conditions (UV exposure versus proteasome inhibition) to shift interaction profiles

  • Employ proximity labeling approaches to identify microenvironment-specific interactions

• Genetic complementation:

  • Express mutant forms targeting specific functions

  • Test whether simultaneous expression of separate mutants restores full functionality

  • Use CRISPR-based approaches for domain-specific mutations rather than complete knockout

These approaches can help delineate the mechanistic details of RAD23B's dual functionality in experimental systems .

What are the latest methodological advances in studying RAD23B interactions and function?

Recent methodological advances have expanded our ability to study RAD23B:

• Immuno-MRM (multiple reaction monitoring):

  • Allows quantitative measurement of RAD23B and its interaction partners

  • Combines immunoaffinity enrichment with targeted mass spectrometry

  • Provides higher specificity than traditional immunoassays

• Advanced imaging approaches:

  • Super-resolution microscopy enables visualization of RAD23B in multiprotein complexes

  • Live-cell imaging with fluorescently tagged RAD23B reveals dynamic interactions

  • FRET/BRET approaches can detect direct protein-protein interactions in living cells

• Single-cell technologies:

  • Flow cytometry analysis provides quantitative measurement of RAD23B at the single-cell level

  • Single-cell RNA-seq combined with protein analysis reveals expression heterogeneity

  • Mass cytometry allows multiplex detection of RAD23B and numerous interaction partners

• Structural biology integration:

  • Antibody epitope mapping combined with structural information

  • Integration of crosslinking mass spectrometry with antibody-based detection

  • Structure-guided antibody development for specific functional domains

These emerging methodologies provide researchers with powerful tools to investigate RAD23B biology at unprecedented resolution and specificity .