DDB2 Antibody

What is DDB2 and why is it important in research?

DDB2 (DNA damage-binding protein 2) is a 427 amino acid protein containing seven WD repeats that belongs to the WD repeat DDB2/WDR76 family. It is ubiquitously expressed throughout the body, with highest expression levels in corneal endothelium and lowest levels in brain tissue. DDB2 primarily localizes in the nucleus and plays a critical role in DNA repair mechanisms. Its importance extends to cancer research, where it has been shown to regulate breast cancer cell growth through negative regulation of SOD2 gene expression. The protein's role as a potential oncogene makes it a promising candidate as a predictive marker in breast cancer research .

What are the key technical specifications of DDB2 antibody?

DDB2 antibody (such as the 30173-1-AP product) typically targets the DDB2 protein in applications such as Western Blot (WB) and ELISA. The antibody shows reactivity with human, mouse, and rat samples, making it versatile for cross-species research. It is commonly available as a rabbit polyclonal IgG, purified through antigen affinity methods. The calculated molecular weight of DDB2 is 48 kDa, while the observed molecular weight in experimental conditions typically ranges from 48-51 kDa. This slight variation may result from post-translational modifications of the target protein .

What samples are most suitable for DDB2 antibody detection?

The DDB2 antibody has been successfully tested against multiple sample types. Positive Western Blot detection has been confirmed in various cell lines including A431, PC-12, HCT 116, HeLa, HepG2, Jurkat, and NIH/3T3 cells. Additionally, the antibody has shown effective detection in mouse kidney and liver tissue samples. This broad range of validated samples makes the antibody suitable for diverse research applications across different biological contexts and experimental models .

How should DDB2 antibody be stored for maximum stability and effectiveness?

DDB2 antibody should be stored at -20°C in its provided buffer, which typically contains PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. Under these conditions, the antibody remains stable for one year after shipment. Notably, aliquoting is unnecessary for -20°C storage, which simplifies laboratory handling. Some preparations (20μl sizes) may contain 0.1% BSA as a stabilizer. Researchers should avoid repeated freeze-thaw cycles to maintain antibody activity and specificity throughout the experimental timeframe .

How can I validate DDB2 antibody specificity in my experimental system?

To validate DDB2 antibody specificity, researchers should consider multiple approaches: (1) Positive controls using cell lines known to express DDB2 (such as HeLa or HCT 116); (2) Negative controls using DDB2 knockdown or knockout samples; (3) Confirmation of the correct molecular weight band (48-51 kDa); (4) Comparison with a second DDB2 antibody targeting a different epitope; and (5) Immunofluorescence co-localization studies to confirm nuclear localization pattern. For key experiments, validation through mass spectrometry identification of the immunoprecipitated protein can provide definitive confirmation of antibody specificity .

How can DDB2 antibody be used to investigate DNA damage repair mechanisms?

DDB2 antibody serves as a crucial tool for examining nucleotide excision repair (NER) pathways, particularly in response to UV radiation damage. Researchers can employ the antibody in chromatin immunoprecipitation (ChIP) assays to examine DDB2 recruitment to sites of DNA damage. Combined with immunofluorescence techniques, researchers can track the temporal and spatial dynamics of DDB2 localization after DNA damage induction. Co-immunoprecipitation (Co-IP) experiments using DDB2 antibody can identify protein complexes formed during repair processes. Additionally, kinetic studies comparing DDB2 binding to damaged DNA sites across different repair-proficient and repair-deficient cell lines can provide mechanistic insights into the early stages of DNA damage recognition .

What is the relationship between DDB2 and CDT2, and how can this be studied experimentally?

DDB2 and CDT2 have an intriguing relationship where CRL4^DDB2 functions as a novel E3 ubiquitin ligase for CDT2. Experimentally, this relationship can be investigated through several approaches: (1) Co-immunoprecipitation assays to confirm DDB2-CDT2 protein interaction; (2) In vivo ubiquitination assays to demonstrate DDB2-mediated ubiquitination of CDT2; (3) Protein stability assays using cycloheximide chase to measure CDT2 half-life in the presence or absence of DDB2; (4) siRNA knockdown of DDB2 followed by Western blot analysis of CDT2 protein levels; and (5) Overexpression studies with DDB2 to observe CDT2 degradation. These experimental approaches can elucidate the molecular mechanisms by which DDB2 regulates CDT2 protein stability .

How does DDB2's regulation of CDT2 impact DNA replication, and what experimental approaches can measure this?

DDB2's regulation of CDT2 has significant implications for DNA replication through indirect regulation of CDT1 protein stability. This process can be experimentally investigated through: (1) Flow cytometry analysis to measure cell cycle progression and detect DNA re-replication events in cells with DDB2 knockdown, CDT2 knockdown, or combined knockdown; (2) Immunofluorescence microscopy to detect nuclear morphology changes and formation of giant nuclei indicative of re-replication; (3) Chromatin fractionation assays to measure MCM2-7 loading onto chromatin following DDB2 manipulation; (4) Protein analysis of pre-replication complex components after DDB2 silencing; and (5) DNA fiber analysis to directly measure replication fork progression. These approaches provide complementary insights into how the DDB2-CDT2-CDT1 regulatory axis controls DNA replication licensing and execution .

What is the prognostic significance of DDB2 expression in different cancer types?

DDB2 functions as a favorable prognostic marker in several cancer types including endometrial cancer, cervical cancer, and breast cancer. This contrasts with CDT2, which typically correlates with poor prognosis. Researchers investigating DDB2 as a prognostic marker should consider: (1) Immunohistochemical analysis of tumor tissue microarrays using validated DDB2 antibodies; (2) Correlation of DDB2 expression levels with patient survival data and clinical parameters; (3) Multivariate analysis to determine if DDB2 is an independent prognostic factor; (4) Comparative analysis of DDB2 and CDT2 expression patterns in the same tumor samples; and (5) Evaluation of DDB2 expression in normal tissue versus tumor tissue. These approaches can establish the clinical relevance of DDB2 as a potential biomarker for disease progression and treatment response .

What experimental systems best model the oncogenic versus tumor-suppressive functions of DDB2?

The dual nature of DDB2 as both potential oncogene and tumor suppressor can be investigated through several experimental systems: (1) Cell line panels representing different cancer types with varying DDB2 expression levels; (2) Isogenic cell line pairs with DDB2 knockout or overexpression; (3) 3D organoid cultures derived from normal and tumor tissues to study DDB2 function in more physiologically relevant contexts; (4) Xenograft models using cells with modified DDB2 expression to assess in vivo tumor growth and metastasis; and (5) Genetically engineered mouse models with tissue-specific DDB2 manipulation. When designing these studies, researchers should carefully consider the tissue context, as DDB2 may exhibit context-dependent functions across different cancer types .

How does DDB2 regulate cancer cell growth, and what experimental approaches can elucidate these mechanisms?

DDB2 regulates cancer cell growth through multiple mechanisms, including negative regulation of SOD2 gene expression in breast cancer cells. To investigate these regulatory pathways, researchers can employ: (1) ChIP-seq analysis to identify genome-wide DDB2 binding sites; (2) RNA-seq following DDB2 manipulation to identify transcriptional targets; (3) Reporter gene assays to validate direct transcriptional regulation; (4) Analysis of cell cycle progression, apoptosis, and senescence after DDB2 knockdown or overexpression; (5) Metabolic profiling to identify DDB2-dependent changes in cellular metabolism; and (6) Analysis of DNA damage response pathways in DDB2-modified cancer cells. These comprehensive approaches can reveal the molecular mechanisms underlying DDB2's role in cancer cell growth regulation .

What are common issues in Western Blot detection of DDB2 and how can they be resolved?

Western Blot detection of DDB2 may encounter several technical challenges:

The recommended dilution range of 1:2000-1:16000 provides flexibility for optimization. For particularly challenging samples, overnight primary antibody incubation at 4°C often improves specific signal detection .

How can I optimize DDB2 antibody performance in different experimental conditions?

Optimizing DDB2 antibody performance requires systematic adjustment of multiple parameters:

• For Western Blot applications:

  • Test a dilution series (1:2000, 1:4000, 1:8000, 1:16000) to identify optimal concentration

  • Vary blocking agents (BSA vs. non-fat milk) as DDB2 detection may be sensitive to blocking conditions

  • Adjust incubation time and temperature to balance signal strength and background

• For immunoprecipitation:

  • Determine optimal antibody-to-lysate ratio for maximum DDB2 pull-down

  • Test different lysis buffers to preserve protein-protein interactions

  • Consider pre-clearing lysates to reduce non-specific binding

• For immunofluorescence:

  • Evaluate different fixation methods (paraformaldehyde vs. methanol)

  • Test antigen retrieval techniques if working with tissue sections

  • Optimize antibody dilutions independently for each application

What controls should be included when studying DDB2-CDT2 interactions?

When investigating DDB2-CDT2 interactions, several critical controls should be included:

• For co-immunoprecipitation studies:

  • Input controls showing expression levels of both proteins

  • IgG control to identify non-specific binding

  • Reciprocal IP (pull-down with CDT2 antibody and probe for DDB2)

  • Conditions with and without proteasome inhibitors

• For ubiquitination assays:

  • Control with proteasome inhibitor alone

  • DDB2 overexpression without ubiquitin co-expression

  • Mutant DDB2 lacking E3 ligase activity

  • Control for specificity using other potential substrates

• For functional studies:

  • Single knockdowns of DDB2 and CDT2 compared to double knockdown

  • Rescue experiments with siRNA-resistant constructs

  • Time-course analyses to establish sequence of molecular events

  • Cell cycle synchronization to determine cell cycle-dependent effects

How is DDB2 involved in the regulation of pre-replication complex assembly?

DDB2 plays a crucial role in pre-replication complex (pre-RC) assembly through its regulation of CDT2 and subsequent effects on CDT1 stability. Experimental evidence indicates that knockdown of DDB2 leads to decreased protein levels of CDT1, which in turn affects the recruitment of MCM2-7 proteins to chromatin. Specifically, silencing DDB2 results in significant reduction of MCM2, MCM3, and MCM7 on chromatin while total MCM protein levels remain unchanged. This suggests that DDB2-mediated degradation of CDT2 through the CRL4^DDB2 ubiquitin ligase is required for stabilizing CDT1, which then facilitates MCM recruitment during pre-RC assembly. This regulatory pathway represents a novel mechanism for DNA replication control that extends beyond DDB2's traditionally studied role in nucleotide excision repair .

What methodological approaches can distinguish between DDB2's roles in DNA repair versus DNA replication?

To distinguish between DDB2's functions in DNA repair versus DNA replication, researchers can implement several sophisticated experimental strategies:

• Temporal analysis using synchronized cell populations to separate replication timing from repair events

• Site-specific DNA damage induction (e.g., laser microirradiation) followed by live-cell imaging of fluorescently tagged DDB2

• Domain-specific mutants of DDB2 that selectively disrupt either repair or replication functions

• ChIP-seq analysis comparing DDB2 binding sites during normal replication versus after DNA damage

• Proteomic analysis of DDB2 interaction partners in different cellular contexts using BioID or APEX proximity labeling

• Single-molecule studies tracking DDB2 dynamics during replication versus repair

• Genetic screens to identify factors that specifically affect either the repair or replication functions of DDB2

How does the CRL4^DDB2 ubiquitin ligase selectivity for CDT2 differ from its activity toward other substrates?

The CRL4^DDB2 ubiquitin ligase complex demonstrates substrate selectivity that appears to function through different mechanisms for different targets. When comparing CRL4^DDB2-mediated degradation of CDT2 versus other known substrates (like XPC, H2A, p27):

• PCNA-dependency: Unlike many CRL4^DDB2 substrates that require PCNA interaction for degradation, DDB2-mediated CDT2 degradation appears to be PCNA-independent. Knockdown of PCNA has negligible effects on CDT2 stability while significantly affecting other substrates like CDT1, p21, and SET8.

• Interaction domains: The study suggests that although both DDB2 and CDT2 contain PIP (PCNA-interacting protein) boxes, the PIP box is dispensable for DDB2-mediated CDT2 degradation, indicating a unique interaction mechanism.

• Downstream effects: DDB2-mediated degradation of CDT2 has differential effects on CDT2's substrates, with strong impacts on CDT1 levels but less pronounced effects on p21 and SET8 levels.

These differences suggest specific structural or contextual requirements for CRL4^DDB2 ubiquitin ligase activity toward CDT2 that distinguish it from other ubiquitination targets .

How do researchers reconcile DDB2's apparently contradictory roles as both oncogene and tumor suppressor?

The seemingly contradictory roles of DDB2 as both oncogene and tumor suppressor represent a significant research question. This paradox can be approached through several investigative strategies:

• Tissue context analysis: Comprehensive studies across multiple tissue types to determine if DDB2 functions differentially depending on tissue origin

• Molecular pathway mapping: Detailed analysis of downstream pathways activated by DDB2 in different cellular contexts

• Interaction partner profiling: Identification of tissue-specific or condition-specific interaction partners that might redirect DDB2 function

• Genetic background analysis: Evaluation of how different genetic contexts (e.g., p53 status) might influence DDB2 function

• Post-translational modification assessment: Investigation of how different modifications of DDB2 might switch its function between oncogenic and tumor-suppressive

• Quantitative threshold analysis: Determination if DDB2 function depends on its expression level, with different outcomes at different concentrations

What explains the differential effects of DDB2 on CDT1, p21, and SET8 stability?

The research indicates an intriguing pattern where DDB2-mediated degradation of CDT2 has varied effects on downstream CDT2 substrates. While CDT1 levels are significantly reduced upon DDB2 silencing, p21 is only slightly decreased, and SET8 appears unaffected. Several hypotheses might explain this differential regulation:

• Substrate affinity differences: CDT2 may have varying binding affinities for its different substrates, resulting in prioritized degradation of CDT1

• Co-factor requirements: Additional co-factors might be necessary for efficient targeting of p21 and SET8 but not for CDT1

• Subcellular compartmentalization: Differential localization of substrates might affect their accessibility to CDT2

• Redundant degradation pathways: Alternative ubiquitin ligases might compensate for CDT2 in targeting p21 and SET8

• Post-translational modifications: Different modifications on substrates might affect their susceptibility to CDT2-mediated degradation

Experimental approaches to test these hypotheses would include subcellular fractionation studies, co-immunoprecipitation with interaction mapping, and targeted inhibition of alternative degradation pathways .