DTX11 Antibody

What is DDX11 and why is it important in molecular biology research?

DDX11, also known as CHLR1 or KRG2, belongs to the DEAD-box protein family, characterized by the conserved Asp-Glu-Ala-Asp motif. It functions primarily as an RNA helicase, playing crucial roles in several cellular processes including RNA secondary structure modifications and assembly of ribosomes and spliceosomes. DDX11 is essential for proper chromosome segregation and embryonic development, with binding capability to both single- and double-stranded DNA. The protein shows high expression in tissues such as testis, thymus, ovary, spleen, and pancreas, highlighting its importance in cellular growth and division. Five isoforms of DDX11 resulting from alternative splicing have been identified, underscoring its complexity and regulatory potential in various biological contexts .

What detection methods are compatible with DDX11 antibodies?

DDX11 Antibody (D-2) has been validated for multiple detection methods, making it versatile for various research applications. The antibody can be used effectively for:

• Western blotting (WB)

• Immunoprecipitation (IP)

• Immunofluorescence (IF)

• Enzyme-linked immunosorbent assay (ELISA)

The antibody is available in multiple formats, including non-conjugated form and various conjugated forms such as agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates to suit different experimental needs .

What is the subcellular localization of DDX11 and how does this affect antibody selection?

DDX11 localizes primarily in the nucleus, which has important implications for antibody selection and experimental design. When performing immunofluorescence or immunohistochemistry, researchers should consider:

• Proper fixation and permeabilization protocols to ensure nuclear access

• Selection of antibody formats that efficiently penetrate the nuclear membrane

• Use of appropriate nuclear counterstains (e.g., DAPI) for co-localization studies

• Optimization of antigen retrieval methods if working with fixed tissues

Understanding the nuclear localization of DDX11 helps explain its functional role in DNA processes and can guide proper experimental design when using anti-DDX11 antibodies .

How can DDX11 antibodies be utilized to study chromosome cohesion defects?

DDX11 plays a critical role in sister chromatid cohesion, making DDX11 antibodies valuable tools for studying cohesion defects. Advanced research applications include:

• Chromatin immunoprecipitation (ChIP) assays to identify DDX11 binding sites on chromosomes

• Proximity ligation assays (PLA) to detect interactions between DDX11 and cohesion complex components

• Immunofluorescence microscopy to visualize cohesion defects in DDX11-depleted cells

• Co-immunoprecipitation experiments to identify novel DDX11 interaction partners

When designing such experiments, researchers should consider cell cycle synchronization methods to enrich for mitotic cells where cohesion defects are most apparent. The DDX11 antibody can be paired with antibodies against known cohesion proteins (e.g., cohesin subunits) to comprehensively map the role of DDX11 in maintaining chromosome integrity .

What are the considerations for using DDX11 antibodies in cancer research applications?

DDX11 has emerging roles in cancer biology, making DDX11 antibodies potentially valuable in oncology research. When employing these antibodies in cancer studies, researchers should consider:

• Expression profiling across cancer cell lines to identify DDX11-high and DDX11-low models

• Correlation of DDX11 expression with clinical parameters and patient outcomes

• Investigation of DDX11's role in DNA repair pathways often dysregulated in cancers

• Analysis of DDX11 expression changes following genotoxic therapies

Research methodologies might include tissue microarray (TMA) analysis, xenograft immunohistochemistry, and flow cytometric assessment of DDX11 in circulating tumor cells. When interpreting results, researchers should be aware that DDX11 expression patterns may vary significantly between cancer types and even between patients with the same cancer diagnosis .

How can multiplexed approaches incorporate DDX11 antibodies to study DNA repair mechanisms?

Advanced multiplexed approaches allow simultaneous detection of DDX11 alongside other DNA repair factors:

• Multi-color immunofluorescence combining DDX11 antibody with antibodies against DNA damage markers (γH2AX, 53BP1)

• Mass cytometry (CyTOF) incorporating metal-conjugated DDX11 antibodies for single-cell analysis

• Imaging mass cytometry for spatial analysis of DDX11 in relation to chromatin territories

• Sequential immunoprecipitation strategies to identify DDX11-containing protein complexes

When designing multiplexed experiments, careful antibody panel design is essential to minimize spectral overlap and cross-reactivity. Controls should include single-stained samples and isotype controls for each antibody in the panel to enable accurate compensation and background correction .

What are the optimal conditions for using DDX11 antibody in Western blotting applications?

For optimal Western blot results with DDX11 antibody:

• Sample preparation:

  • Use RIPA or NP-40 buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if studying DDX11 phosphorylation

  • Sonicate samples to shear DNA and ensure complete protein extraction

• Gel electrophoresis:

  • Use 8-10% SDS-PAGE gels for optimal separation (DDX11 is ~108 kDa)

  • Load 20-40 μg of total protein per lane

  • Include positive control lysates from cells known to express DDX11

• Transfer and detection:

  • Transfer at 100V for 90 minutes or 30V overnight at 4°C

  • Block with 5% non-fat milk or BSA in TBST

  • Use DDX11 antibody at 1:500-1:1000 dilution

  • Incubate with primary antibody overnight at 4°C

  • Wash thoroughly (4 x 5 minutes) before secondary antibody addition

Expected results should show a specific band at approximately 108 kDa. Alternative splicing of DDX11 may result in additional bands of different molecular weights, which should be verified through knockdown experiments .

What are the recommended protocols for optimizing immunofluorescence with DDX11 antibodies?

For successful immunofluorescence using DDX11 antibodies:

• Fixation methods:

  • 4% paraformaldehyde (10 minutes at room temperature) for structural preservation

  • Methanol fixation (-20°C for 10 minutes) for nuclear antigen access

  • Avoid over-fixation which can mask epitopes

• Permeabilization:

  • 0.1-0.5% Triton X-100 for 5-10 minutes

  • Alternative: 0.1% Saponin if milder permeabilization is preferred

• Blocking and antibody incubation:

  • Block with 5% normal serum from the species of the secondary antibody

  • Use DDX11 antibody at 1:100-1:500 dilution

  • Incubate primary antibody for 1-2 hours at room temperature or overnight at 4°C

  • Use secondary antibodies at 1:500-1:1000 dilution

• Counterstaining:

  • DAPI for nuclear visualization

  • Consider additional markers for nucleoli or specific nuclear compartments

Expect predominantly nuclear staining with possible nucleolar enrichment. Include appropriate controls including secondary-only samples and known DDX11-negative cell types .

How should researchers approach epitope mapping when working with DDX11 antibodies?

Epitope mapping for DDX11 antibodies requires systematic approach:

• Bioinformatic analysis:

  • Predict antigenic determinants using tools like BepiPred or ABCpred

  • Analyze evolutionary conservation of epitope regions

  • Assess potential post-translational modifications that might affect epitope recognition

• Experimental mapping strategies:

  • Express truncated DDX11 fragments to narrow down binding regions

  • Use peptide arrays spanning the DDX11 sequence

  • Perform competition assays with synthetic peptides

  • Consider hydrogen-deuterium exchange mass spectrometry for conformational epitopes

• Validation approaches:

  • Confirm epitope accessibility in native protein using structural modeling

  • Verify epitope conservation across species if cross-reactivity is desired

  • Test antibody recognition under different denaturing conditions

Understanding the specific epitope recognized by a DDX11 antibody can explain differential results between applications (e.g., why an antibody works for Western blot but not IP) and guide appropriate experimental design .

How can researchers troubleshoot non-specific binding when using DDX11 antibodies?

Non-specific binding with DDX11 antibodies can be addressed through:

• Optimization strategies:

  • Titrate antibody concentration to find optimal signal-to-noise ratio

  • Increase blocking stringency (5-10% blocking agent)

  • Add 0.1-0.5% Tween-20 to antibody dilution buffer

  • Pre-adsorb antibody with cell/tissue lysate from DDX11-negative samples

• Cross-reactivity assessment:

  • Perform knockdown/knockout validation

  • Test antibody specificity using overexpression systems

  • Compare staining patterns with multiple DDX11 antibodies targeting different epitopes

• Background reduction techniques:

  • For IF: Include 0.1-0.3M glycine to quench aldehyde groups after fixation

  • For WB: Increase washing duration and number of washes

  • For IP: Use protein-specific rather than general protein A/G beads

The DDX11 antibody may cross-react with other DEAD-box family members due to conserved domains. Careful validation using genetic approaches (siRNA, CRISPR) is recommended to confirm specificity, particularly in new cell types or tissues .

What factors should be considered when interpreting DDX11 expression data across different cell types?

When analyzing DDX11 expression across cell types:

• Cell cycle considerations:

  • DDX11 expression may fluctuate throughout the cell cycle

  • Synchronize cells or use cell cycle markers for accurate comparison

  • Normalize data to appropriate housekeeping genes based on cell type

• Tissue-specific expression patterns:

  • Consider baseline expression differences (higher in testis, thymus, ovary, spleen, pancreas)

  • Interpret results in context of tissue-specific splice variants

  • Account for potential differences in post-translational modifications

• Technical considerations:

  • Use multiple detection methods to confirm expression patterns

  • Include quantification methods with appropriate statistical analysis

  • Consider antibody affinities for different DDX11 isoforms

Researchers should be aware that DDX11 is not present on normal fibroblast cell lines or tumor cell lines of epithelial or neuronal origins, which can serve as negative controls in expression studies .

How can researchers distinguish between the five isoforms of DDX11 in experimental systems?

Distinguishing between DDX11 isoforms requires targeted approaches:

• Isoform-specific detection strategies:

  • Design PCR primers spanning unique exon junctions

  • Use isoform-specific antibodies (if available)

  • Employ mass spectrometry to identify isoform-specific peptides

• Experimental design considerations:

  • Include controls expressing single isoforms for size comparison

  • Use 2D gel electrophoresis to separate isoforms by both size and charge

  • Consider native protein electrophoresis to preserve structural differences

• Analysis methods:

  • Create a reference table of expected molecular weights for each isoform

  • Use phosphorylation-specific antibodies if isoforms differ in phosphorylation sites

  • Employ computational tools to predict isoform-specific functions based on domain preservation

Researchers should be cautious when interpreting DDX11 antibody results, as commercial antibodies may have differential reactivity toward the five isoforms. Validation studies involving recombinant expression of each isoform can help establish the detection profile of the antibody being used .

How can ChIP-seq be optimized using DDX11 antibodies to study chromatin interactions?

Optimizing ChIP-seq with DDX11 antibodies requires:

• Crosslinking optimization:

  • Test different formaldehyde concentrations (0.5-2%)

  • Consider dual crosslinking with additional agents (DSG, EGS)

  • Optimize crosslinking time (5-20 minutes) based on cell type

• Sonication parameters:

  • Aim for 200-500 bp fragments

  • Validate sonication efficiency by gel electrophoresis

  • Consider enzymatic fragmentation alternatives

• Immunoprecipitation conditions:

  • Increase antibody concentration for ChIP-grade performance

  • Extend incubation time (overnight at 4°C)

  • Include appropriate controls (IgG, input, positive locus control)

• Data analysis considerations:

  • Use appropriate peak-calling algorithms

  • Compare DDX11 binding sites with known DNA repair and replication origins

  • Integrate with expression data to correlate binding with function

DDX11's ability to bind both single- and double-stranded DNA makes it important to establish stringent controls and validation steps for ChIP-seq experiments to differentiate specific from non-specific binding events .

What considerations should be made when designing CRISPR-based studies targeting DDX11?

When designing CRISPR studies for DDX11:

• Guide RNA design strategies:

  • Target conserved exons present in all isoforms for complete knockout

  • Target specific exons for isoform-selective studies

  • Avoid regions with high homology to other DEAD-box helicases

  • Consider targeting regulatory regions for expression modulation

• Phenotypic analysis approaches:

  • Monitor cell proliferation and cell cycle profiles

  • Assess chromosome segregation errors using live-cell imaging

  • Evaluate DNA damage accumulation using γH2AX foci

  • Test sensitivity to DNA damaging agents

• Rescue experiment design:

  • Create rescue constructs resistant to guide RNA targeting

  • Generate domain mutants to assess specific functional requirements

  • Consider inducible systems for temporal control of DDX11 expression

• Control considerations:

  • Include non-targeting guide RNA controls

  • Generate multiple independent clones to account for clonal variation

  • Validate knockout efficiency using both protein and mRNA detection methods

Given DDX11's essential role in embryonic development, complete knockout may be lethal in some cell types, necessitating the use of conditional or inducible CRISPR systems .