DDB1 Monoclonal Antibody

What is DDB1 and what is its primary function in cells?

DDB1 is a 127 kDa protein that serves as a crucial component of the UV-damaged DNA-binding protein complex (UV-DDB). It binds to DDB2 to form this complex, which recognizes UV-induced DNA damage and recruits proteins of the nucleotide excision repair pathway to initiate DNA repair . DDB1 is highly conserved across mammals, insects, worms, and plants, indicating its evolutionary importance .

In conjunction with CUL4A and ROC-1, the UV-DDB complex forms an E3 ubiquitin ligase that recognizes various DNA lesions including cyclobutane pyrimidine dimers, 6-4 photoproducts, apurinic sites, and short mismatches . This complex plays a critical role in the polyubiquitination of components within the nucleotide excision repair pathway.

What applications are DDB1 monoclonal antibodies suitable for?

DDB1 monoclonal antibodies have been validated for several research applications:

For optimal results, it is recommended to titrate the antibody in each testing system, as optimal concentrations may be sample-dependent .

What are the recommended storage conditions for DDB1 antibodies?

For long-term stability, DDB1 monoclonal antibodies should be stored at -20°C, where they remain stable for one year after shipment . The antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Aliquoting is generally unnecessary for -20°C storage, though some preparations (20μl sizes) may contain 0.1% BSA . It's important to avoid repeated freeze-thaw cycles to maintain antibody activity.

What are the critical considerations for DDB1 detection in Western blotting?

When detecting DDB1 via Western blotting, researchers should consider:

• Protein Size: DDB1 has a calculated molecular weight of 127 kDa (1140 amino acids) , which should be confirmed on your blot.

• Antibody Dilution: For most samples, a dilution range of 1:500-1:2000 is recommended , but optimization might be necessary depending on your specific sample.

• Positive Controls: Validated cell lines for positive control include HeLa, MCF-7, A549, HEK-293, Jurkat, HSC-T6, PC-12, NIH/3T3, and RAW 264.7 cells .

• Species Reactivity: Check antibody reactivity with your species of interest. The antibody discussed in the search results shows reactivity with human, mouse, and rat samples .

• Validation Strategy: Consider using knockout/knockdown controls as implemented in published studies to confirm specificity .

What protocols are recommended for immunohistochemical detection of DDB1?

For optimal IHC detection of DDB1:

• Antigen Retrieval: Use TE buffer at pH 9.0 as the primary recommendation. Alternatively, citrate buffer at pH 6.0 can be used .

• Antibody Dilution: A dilution range of 1:500-1:2000 is recommended for IHC applications .

• Validated Tissues: Human appendicitis tissue and human colon cancer tissue have been successfully used for IHC validation .

• Detection System: Choose an appropriate secondary antibody and visualization system compatible with mouse IgG2b, as the mentioned antibody is of this isotype .

• Controls: Include positive and negative controls to validate staining specificity.

How can researchers study the DDB1-AMBRA1 E3 ligase complex structure and function?

Recent structural studies have provided insights into the DDB1-AMBRA1 complex, revealing important mechanisms about this E3 ligase receptor:

• Structural Analysis: The cryo-EM structure of the human DDB1-AMBRA1 WD40 complex shows that parts of the N- and C-terminal structural regions in AMBRA1 fold together into a highly dynamic WD40 domain . This approach revealed how DDB1 engages with AMBRA1 to create a binding scaffold for substrate recruitment.

• Functional Domains: The N-terminal helix-loop-helix motif of AMBRA1 is essential for DDB1 binding, and point mutations within this region abolish their interaction .

• Substrate Interactions: The complex is involved in the ubiquitination of Cyclin D1. Researchers can study this by using DDB1 binding-defective AMBRA1 mutants, which prevent ubiquitination of Cyclin D1 in vitro and increase cell cycle progression .

• HDX-MS Analysis: This technique has been applied to show that DDB1 binding stabilizes distinct AMBRA1 regions, allowing for structural determination .

• Physiological Relevance: This complex acts as a hub involved in various processes including autophagy and cell cycle regulation, making it valuable for investigating cellular pathways intersecting these processes .

What methods can be used to investigate DDB1's role in the nucleotide excision repair pathway?

To study DDB1's function in nucleotide excision repair (NER):

• UV-Damage Assays: Since DDB1 is involved in recognizing UV-induced DNA damage, researchers can design experiments with controlled UV exposure followed by analysis of repair kinetics.

• Protein Complex Analysis: Investigate the interactions between DDB1, DDB2, CUL4A, and ROC-1 to understand how the E3 ubiquitin ligase complex forms and functions in the NER pathway .

• Histone Ubiquitination Studies: The DDB1-DDB2-CUL4A-ROC1 E3 ubiquitin-protein ligase complex ubiquitinates histones H2A, H3, and H4 at DNA damage sites, potentially facilitating their removal from the nucleosome to promote DNA repair . Researchers can use chromatin immunoprecipitation techniques to study these modifications.

• Mutation Analysis: Create or study mutations in DDB1 and analyze their effects on DNA repair capacity. Unlike DDB2, lesions in DDB1 have not been identified in xeroderma pigmentosum complementation group E (XP-E) patients, making this an area of potential research interest .

• Substrate Targeting Studies: DDB1 functions to recruit substrate-specific targeting subunits (DCAFs or CDWs) to CUL4-RING E3 ubiquitin-protein ligase complexes . Investigating these interactions can provide insights into its role in NER.

What are common issues in DDB1 antibody applications and how can they be resolved?

When working with DDB1 monoclonal antibodies, researchers might encounter several challenges:

• Background Signals: High background can interfere with specific detection. Optimize blocking conditions (typically using BSA or non-fat milk) and increase washing steps. For Western blotting, consider using PVDF membranes which may provide better signal-to-noise ratios for large proteins like DDB1.

• Antibody Specificity: Confirm specificity using appropriate controls, including knockdown/knockout samples as demonstrated in published studies .

• Detection Sensitivity: If signal is weak in Western blotting, consider increasing protein loading, extending primary antibody incubation time, or using enhanced chemiluminescence (ECL) substrates with higher sensitivity.

• Cross-Reactivity: Although the described antibody shows reactivity with human, mouse, and rat samples , cross-reactivity should be verified when working with other species or unusual sample types.

• IHC Optimization: If IHC staining is suboptimal, try both recommended antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0) to determine which works best for your tissue samples.

How can researchers differentiate between DDB1's multiple cellular functions in experimental designs?

DDB1 participates in various cellular processes, including DNA repair, cell cycle regulation, and autophagy. To distinguish between these functions:

• Function-Specific Binding Partners: Co-immunoprecipitation with function-specific binding partners (e.g., DDB2 for DNA repair, AMBRA1 for autophagy/cell cycle) can help isolate specific complexes .

• Cellular Compartmentalization: Subcellular fractionation followed by immunoblotting can help determine the localization of DDB1 in different cellular compartments, which may correlate with specific functions.

• Specific Substrate Analysis: For example, studying Cyclin D1 ubiquitination would focus on DDB1's cell cycle regulatory function , while analyzing histone modifications would emphasize its DNA repair role .

• Pathway-Specific Inhibitors: Use inhibitors of specific pathways (DNA repair, autophagy, ubiquitin-proteasome system) to isolate the function being studied.

• Temporal Analysis: Some functions may be more prominent at different cell cycle stages or following specific cellular stresses (e.g., UV damage for DNA repair function), allowing temporal separation of activities.

What are the emerging roles of DDB1 beyond classical DNA repair functions?

Recent research has expanded our understanding of DDB1's functions beyond its classical role in DNA repair:

• Cell Cycle Regulation: The DDB1-AMBRA1 complex affects cell cycle progression through regulation of Cyclin D1 levels. Loss of AMBRA1 leads to accumulation of Cyclin D1 and decreased sensitivity to CDK4/6 inhibitors, resulting in increased tumorigenic potential .

• Autophagy Coordination: AMBRA1 is a central hub for autophagy coordination, and its interaction with DDB1 suggests DDB1's involvement in this process as well .

• E3 Ligase Substrate Specificity: DDB1 functions to recruit substrate-specific targeting subunits to CUL4-RING E3 ubiquitin-protein ligase complexes, determining the functional specificity of these complexes .

• Epigenetic Regulation: Through its role in histone ubiquitination, DDB1 may participate in broader epigenetic regulatory mechanisms beyond just facilitating DNA repair .

• Potential Therapeutic Target: Understanding DDB1's multiple roles could inform the development of targeted therapies for conditions involving dysregulation of these processes, including cancer.

What technologies are advancing the study of DDB1-associated complexes?

Several cutting-edge technologies are enhancing our understanding of DDB1 biology:

• Cryo-Electron Microscopy: This technique has been pivotal in determining the structure of the DDB1-AMBRA1 WD40 complex, revealing crucial insights into how these proteins interact .

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): This approach has shown how DDB1 binding stabilizes distinct AMBRA1 regions, contributing to our understanding of the complex's structure and dynamics .

• CRISPR/Cas9 Gene Editing: Allows for precise modification of DDB1 or its interaction partners to study functional consequences in cellular systems.

• Proximity Labeling Techniques: Methods like BioID or APEX can identify proteins in close proximity to DDB1 in living cells, helping discover new interaction partners and functions.

• Live-Cell Imaging: Advanced microscopy techniques enable visualization of DDB1 dynamics in response to cellular stresses like DNA damage.