DDB1 Antibody, Biotin conjugated

What is DDB1 and what are its primary cellular functions?

DDB1 (Damage-specific DNA binding protein 1) is a 127 kDa protein that serves dual critical functions in cellular processes:

• DNA Repair Role: Core component of the UV-DDB complex (UV-damaged DNA-binding protein complex), which recognizes UV-induced DNA damage and recruits proteins of the nucleotide excision repair pathway (NER) to initiate DNA repair .

• Ubiquitination Role: Functions as a component of numerous distinct DCX (DDB1-CUL4-X-box) E3 ubiquitin-protein ligase complexes that mediate the ubiquitination and subsequent proteasomal degradation of target proteins .

The UV-DDB complex preferentially binds to several types of DNA damage:

• Cyclobutane pyrimidine dimers (CPD)

• 6-4 photoproducts (6-4 PP)

• Apurinic sites

• Short mismatches

DDB1 is also known by several alternative names: XAP1, XPCe, DDBa, DNA damage-binding protein 1, and XPE-BF .

What are the typical applications for DDB1 Antibody, Biotin conjugated?

The biotin-conjugated DDB1 antibody is validated for multiple research applications in human samples:

These applications are particularly valuable for:

• Visualization of DDB1 protein localization in fixed cells and tissues

• Studying DNA damage response pathways

• Investigating ubiquitination mechanisms

• Analyzing protein-protein interactions in the context of DNA repair complexes

The biotin conjugation provides enhanced sensitivity through signal amplification using streptavidin detection systems, which is especially beneficial for detecting proteins expressed at low levels or in specific subcellular compartments .

What are the recommended protocols for using DDB1 Antibody, Biotin conjugated in immunofluorescence studies?

For optimal results in immunofluorescence studies using biotin-conjugated DDB1 antibody:

Sample Preparation:

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.2% Triton X-100 in PBS for 5 minutes

• Block with 5% normal serum (from the same species as the secondary antibody) for 1 hour

Staining Protocol:

• Incubate with biotin-conjugated DDB1 antibody (optimized dilution, typically starting at 1:50-1:200)

• Wash 3× with PBS (5 minutes each)

• Incubate with streptavidin-conjugated fluorophore (e.g., streptavidin-Alexa Fluor 488/594/647)

• Counterstain nuclei with DAPI

• Mount with anti-fade mounting medium

Special Considerations:

• For DDB1 detection in the context of DNA damage, treating cells with UV radiation (10-20 J/m²) 1-6 hours before fixation can enhance visualization of repair complexes

• Nuclear extraction procedures may be necessary to observe specific binding patterns, as DDB1 functions in both nuclear and cytoplasmic compartments

How should I validate the specificity of DDB1 Antibody, Biotin conjugated in my experimental system?

Comprehensive validation should include multiple complementary approaches:

1. Positive and Negative Controls:

• Positive Controls: Use cell lines known to express DDB1 (HeLa, MCF-7, A549, HEK-293, Jurkat)

• Negative Controls: Include secondary-only controls and isotype controls

2. Knockdown/Knockout Validation:

• Implement siRNA/shRNA knockdown of DDB1

• Use CRISPR-Cas9 DDB1 knockout cells if available

• Compare staining patterns between wild-type and KD/KO samples

3. Peptide Competition Assay:

• Pre-incubate the antibody with excess DDB1 immunizing peptide

• Compare staining with and without peptide competition

• Specific signals should be significantly reduced after peptide competition

4. Cross-Validation with Other Antibodies:

• Compare results with non-biotin conjugated DDB1 antibodies (e.g., mouse monoclonal 66010-1-Ig or rabbit polyclonal 11380-1-AP)

• Validate using antibodies targeting different epitopes of DDB1

5. Western Blot Confirmation:

• Confirm specificity by Western blot showing a single band at 127 kDa

• Recommended dilutions for Western blot range from 1:500-1:2000 for most DDB1 antibodies

How can DDB1 Antibody, Biotin conjugated be utilized for studying the interaction between DDB1 and binding partners in the UV-DDB complex?

The biotin-conjugated DDB1 antibody enables sophisticated approaches for studying protein interactions:

1. Sequential Chromatin Immunoprecipitation (ChIP-reChIP):

• First ChIP: Use biotin-conjugated DDB1 antibody to pull down DDB1-associated chromatin

• Second ChIP: Target potential binding partners (e.g., DDB2, CUL4A, CUL4B)

• This approach identifies genomic regions where both proteins co-localize

2. Proximity Ligation Assay (PLA):

• Use biotin-conjugated DDB1 antibody with antibodies against suspected binding partners

• PLA produces fluorescent spots only when proteins are in close proximity (<40 nm)

• Quantify interactions under different conditions (e.g., UV exposure, drug treatments)

3. Co-Immunoprecipitation with Streptavidin Pull-down:

• Cross-link protein complexes with formaldehyde or DSP

• Immunoprecipitate using streptavidin beads to capture biotin-DDB1 antibody complexes

• Analyze by Western blot or mass spectrometry to identify interacting partners

4. Structural Analysis of DDB1 Complexes:
The structure of DDB1 consists of three domains that facilitate different interactions:

• N-terminal domain (NTD): Forms a seven-stranded β-sheet (residues 1-185)

• Helical bundle domain (HBD): Contains 7 α-helices (residues 186-317) involved in DDB1 binding

• C-terminal domain (CTD): Composed of 8 β-sheets (residues 318-445)

DDB1 attaches to binding partners through a cavity between the BPA and BPC propellers of its three WD40 β-propellers arranged in a triangular fashion .

What are the methodological considerations for using DDB1 Antibody, Biotin conjugated in dual or multi-color immunohistochemistry?

1. Signal Separation and Cross-Reactivity:

• When using biotin-conjugated DDB1 antibody with other antibodies, ensure complete blocking of endogenous biotin using biotin/avidin blocking kits

• For multi-color IHC, use tyramide signal amplification systems that allow sequential detection of multiple antigens

2. Protocol Optimization for Multi-color IHC:

• Antigen Retrieval: Use TE buffer pH 9.0 as primary option for DDB1 detection

• Alternative Method: Citrate buffer pH 6.0 can be used as an alternative

• Dilution Range: Start with 1:50-1:500 for IHC applications and optimize based on tissue type

3. Sequential Detection Strategy:

• Detect the biotin-conjugated DDB1 antibody first using streptavidin-HRP and a chromogen

• Perform multiple rounds of microwave treatment to strip previous antibodies

• Continue with detection of additional targets using different chromogens

• Document results at each stage through multi-spectral imaging

4. Tissue-Specific Considerations:
The DDB1 antibody has been successfully validated in:

• Human colon cancer tissue

• Human appendicitis tissue

• Human kidney tissue

• Human placenta tissue

• Human brain tissue

Each tissue may require specific optimization of antigen retrieval conditions and antibody dilutions.

What are common issues when using DDB1 Antibody, Biotin conjugated and how can they be resolved?

1. High Background Signal:

2. Weak or No Signal:

3. Unexpected Subcellular Localization:

4. Cross-reactivity Issues:

How can I optimize DDB1 Antibody, Biotin conjugated for studying UV-induced DNA damage repair pathways?

1. Experimental Design for UV Damage Studies:

2. Advanced Visualization Techniques:

• Super-resolution Microscopy: Use structured illumination or STORM microscopy with biotin-streptavidin detection systems for nanoscale visualization of repair foci

• Live-cell Imaging: Combine with fluorescently tagged repair factors to study kinetics

• FRAP Analysis: Fluorescence recovery after photobleaching to study mobility and binding dynamics

3. Functional Assays with DDB1 Antibody:

• Chromatin Association: Study time-dependent association of DDB1 with chromatin after UV damage

• Repair Kinetics: Combine with antibodies against CPD or 6-4PP to measure damage removal rates

• Ubiquitination Analysis: Pair with ubiquitin antibodies to study histone modifications at damage sites

4. DDB1 Complex Formation Analysis:

The DDB1 antibody can be used to study formation of key complexes:

• DDB1-DDB2 (UV-DDB complex)

• DDB1-CUL4A/B-RBX1 (E3 ligase complexes)

• DDB1-CRBN (immunomodulatory drug target)

Each complex has distinct functions in DNA repair and protein ubiquitination that can be studied through co-localization or co-immunoprecipitation approaches.

How can DDB1 Antibody, Biotin conjugated be applied in studying the role of DDB1 in DNA methylation regulation?

Recent research has implicated DDB1 in DNA methylation regulation, expanding its known functions beyond DNA repair:

1. Methodological Approaches:

• DNA Methylation Analysis: Combine DDB1 ChIP with bisulfite sequencing to correlate DDB1 binding with methylation states

• Methylation-specific PCR: Assess methylation changes at specific loci after DDB1 knockdown

• Co-localization Studies: Examine co-localization of DDB1 with DNA methyltransferases (DNMTs) using the biotin-conjugated antibody

2. Research Model Systems:

• Neurospora Model: Studies in Neurospora have shown that Cul4 and DDB1 are essential for DNA methylation, suggesting a conserved role

• Mammalian Cell Models: Explore similar functions in mammalian systems using the biotin-conjugated DDB1 antibody

3. Experimental Design Considerations:

• Generate antisera against DDB1 following protocols that use GST-DDB1 fusion proteins containing specific amino acid regions (e.g., aa 1056-1159)

• Compare results with commercial biotin-conjugated antibodies to validate findings

• Design methylation-specific assays that target regions affected by DDB1 knockout

What are the considerations for using DDB1 Antibody, Biotin conjugated in studying the DDB1-CRBN E3 ubiquitin ligase complex in the context of immunomodulatory drug research?

The DDB1-CRBN E3 ubiquitin ligase complex has emerged as a critical target for immunomodulatory drugs such as thalidomide and its derivatives:

1. Structural Considerations for Antibody Selection:

• The DDB1-CRBN interaction involves specific domains:

  • DDB1's BPA and BPC propellers form a cavity where CRBN attaches

  • CRBN's helical bundle domain (HBD, residues 186-317) mediates attachment to DDB1

• Ensure the biotin-conjugated antibody's epitope doesn't interfere with these interaction regions

2. Methodological Approaches:

• Protein Microarray Analyses: On-chip ubiquitination assays using CRL4^CRBN in the presence of E1 (Uba1), E2 (UbcH5a), and biotin-ubiquitin can identify novel substrates

• Drug Binding Studies: Use the biotin-conjugated DDB1 antibody to study how immunomodulatory drugs affect complex formation

• Substrate Identification: Combine with mass spectrometry to identify novel substrates

3. Technical Considerations:

• Native Complex Preservation: Use mild lysis conditions (e.g., 0.1% NP-40) to maintain intact complexes

• Drug Treatment Protocols: Include positive controls with known immunomodulatory drugs (thalidomide, lenalidomide)

• Quantification: Implement quantitative approaches (like AQUA peptides) for measuring complex abundance

4. Research Applications:

• Mapping the binding sites of therapeutic compounds on the DDB1-CRBN complex

• Screening novel compounds that modulate DDB1-CRBN function

• Identifying patient-specific biomarkers for response to immunomodulatory drugs