DDX47 Antibody, Biotin conjugated

What is DDX47 and what cellular functions does it perform?

DDX47 (DEAD box protein 47) is a member of the DEAD box protein family, characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD). It functions as an ATP-dependent RNA helicase involved in altering RNA secondary structures . DDX47 plays crucial roles in:

• Ribosome biogenesis - required for efficient pre-rRNA processing

• mRNA splicing - associates with pre-rRNA precursors

• Apoptosis regulation - interacts with apoptotic pathways

• R-loop stability maintenance - depletion disrupts normal RNAPII functioning

• Nucleolar processes - localizes primarily to the nucleolus but can shuttle between nucleus and cytoplasm

Interestingly, DDX47 has significant diagnostic value in various clinical scenarios, particularly in patients with common infections, sepsis, and septic shock, where its reduced expression correlates with immune suppression .

What is the significance of biotin conjugation for DDX47 antibodies?

Biotin conjugation provides several methodological advantages for antibody-based detection of DDX47:

• Enhanced signal sensitivity: The biotin-(strept)avidin system offers extraordinary binding affinity (K_D 10^-14-10^-15), which is 10³-10⁶ times higher than typical antigen-antibody interactions . This enables:

  • Amplification of weak signals

  • Detection of very low DDX47 concentrations

  • Reduced number of experimental steps

• System stability: Biotin conjugation creates detection systems that remain stable despite:

  • Temperature and pH extremes

  • Exposure to proteolytic enzymes

  • Harsh organic reagents and denaturing conditions

• Versatile detection options: Biotin-conjugated DDX47 antibodies can be paired with various streptavidin-coupled detection systems (fluorophores, enzymes, nanoparticles), allowing flexible experimental design across multiple techniques .

• Improved targeting in certain contexts: Biotin transporters (sodium-dependent multivitamin transporters/SMVT) are overexpressed in various aggressive cancer cell lines, potentially allowing preferential targeting of DDX47 in these contexts .

How does biotinylated DDX47 antibody reactivity compare across different species?

Reactivity profiles vary significantly among commercially available DDX47 antibodies. Based on the examined sources, the following species reactivity has been documented:

Researchers should validate cross-reactivity experimentally when working with non-human species, particularly those with predicted rather than confirmed reactivity .

What are the optimal protocols for using biotinylated DDX47 antibody in Western blotting?

Western blotting with biotinylated DDX47 antibody requires specific methodological considerations:

• Sample preparation:

  • Use NETN lysis buffer for optimal protein extraction

  • Load 15-50 μg of whole cell lysate per lane

  • Include positive control lysates from cells with known DDX47 expression (HeLa, HEK-293T, NIH/3T3)

• Dilution optimization:

  • Starting dilution of 1:1000-1:8000 for unconjugated antibodies

  • For biotin-conjugated antibody, initial testing at 1:500 followed by titration

  • For detection, use streptavidin-HRP (typically 1:2000-1:5000)

• Blocking considerations:

  • Use casein-based blockers rather than BSA to avoid endogenous biotin interference

  • Add avidin (10-50 μg/ml) to blocking buffer to sequester endogenous biotin

• Expected results:

  • Primary band at approximately 48-50 kDa

  • Use ECL detection with initial exposure time of 3 minutes

  • For weak signals, employ streptavidin-based signal amplification systems

How can I optimize immunofluorescence protocols using biotinylated DDX47 antibody?

For effective immunofluorescence with biotinylated DDX47 antibody:

• Fixation and permeabilization:

  • 4% paraformaldehyde fixation (10 minutes at room temperature)

  • Permeabilize with 0.1-0.5% Triton X-100 (5-10 minutes)

  • For nucleolar DDX47 detection, extended permeabilization may be necessary

• Blocking and antibody incubation:

  • Block with 5-10% normal serum from species unrelated to primary antibody

  • Include avidin (10-50 μg/ml) in blocking solution to reduce background

  • Apply biotinylated DDX47 antibody at 1:500-1:2000 dilution

  • Incubate overnight at 4°C for optimal signal-to-noise ratio

• Detection system:

  • Use fluorophore-conjugated streptavidin (e.g., Streptavidin-Alexa Fluor)

  • Counterstain with DAPI to identify nuclei/nucleoli

  • Include RNase treatment controls to verify RNA-dependent localization

• Imaging considerations:

  • Expected primarily nucleolar localization with some nucleoplasmic signal

  • Use confocal microscopy for precise subcellular localization

  • Consider co-staining with nucleolar markers (fibrillarin, nucleolin) for confirmation

What validation steps ensure specificity when using biotin-conjugated DDX47 antibody?

Thorough validation is critical for ensuring experimental reliability:

• Positive and negative controls:

  • Use cell lines with known DDX47 expression (HeLa, HEK-293T, NIH/3T3)

  • Include knockout/knockdown controls where possible

  • Employ peptide competition assays with immunizing peptide

• Cross-validation methods:

  • Compare results across multiple detection techniques (WB, IF, IHC)

  • Use alternative DDX47 antibodies targeting different epitopes

  • Verify with non-biotin conjugated antibodies against the same target

• Specificity controls for biotin system:

  • Include secondary-only and streptavidin-only controls

  • Perform blocking of endogenous biotin using avidin

  • Test for cross-reactivity with other DEAD box family members

• Functional validation:

  • Correlate antibody signal with known DDX47 functions

  • Verify nucleolar localization pattern

  • Confirm expected molecular weight (48-50 kDa)

How can biotinylated DDX47 antibody be used to investigate its role in RNA processing?

To explore DDX47's function in RNA metabolism:

• RNA immunoprecipitation (RIP) assays:

  • Use biotinylated DDX47 antibody to pull down DDX47-RNA complexes

  • Employ streptavidin beads for efficient capture

  • Analyze associated RNAs by RT-PCR or sequencing

  • Control for non-specific RNA binding with IgG controls

• Combined protein-RNA visualization:

  • Perform RNA-FISH followed by immunofluorescence with biotinylated DDX47 antibody

  • Use different fluorophore-streptavidin conjugates to distinguish RNA species from protein

  • Analyze co-localization at ribosomal DNA loci and pre-rRNA processing sites

• In situ RNA helicase activity assays:

  • Design fluorescence-based reporter systems with RNA structural elements

  • Monitor structural changes upon DDX47 recruitment

  • Use biotinylated DDX47 antibody to confirm enzyme localization

• R-loop analysis:

  • Combine DDX47 detection with S9.6 antibody (R-loop marker)

  • Assess R-loop accumulation upon DDX47 depletion

  • Correlate DDX47 localization with sites of transcription-replication conflict

What is DDX47's potential as a diagnostic biomarker and how can biotinylated antibodies facilitate its assessment?

DDX47 shows promising diagnostic applications in several contexts:

• Sepsis and infection biomarker:

  • DDX47 expression levels significantly differentiate between healthy individuals and patients with common infections, sepsis, and septic shock

  • Expression remains consistent regardless of the infection site, enhancing clinical utility

  • Biotinylated antibodies enable highly sensitive detection in blood samples

• Cancer diagnostics and prognostics:

  • Poor prognostic indicator for renal cell carcinoma

  • Diagnostic biomarker for breast cancer and chronic myeloid leukemia

  • Biotin-conjugated antibodies can leverage increased biotin receptor expression in cancer cells for enhanced detection specificity

• Implementation methodologies:

  • Develop sandwich ELISA using biotinylated DDX47 antibody and streptavidin-HRP

  • Create multiplex detection systems combining DDX47 with other biomarkers

  • Employ Bridged Avidin-Biotin (BRAB) techniques for enhanced sensitivity

  • Design point-of-care diagnostic platforms utilizing the robust biotin-streptavidin system

• Translational research applications:

  • Monitor DDX47 expression during disease progression

  • Correlate with other immune suppression markers like GATA3

  • Assess response to therapeutic interventions

What are the common troubleshooting challenges when using biotinylated DDX47 antibodies?

Researchers should be prepared to address these frequent technical issues:

• High background in tissues and cells:

  • Cause: Endogenous biotin in samples (particularly prevalent in liver, kidney, and adipose tissue)

  • Solution: Pre-block with avidin/streptavidin before adding biotinylated antibody

  • Alternative: Use avidin-biotin blocking kit prior to antibody incubation

• Inconsistent signal strength:

  • Cause: Variable DDX47 expression based on cell cycle stage or stress conditions

  • Solution: Synchronize cells or standardize stress conditions

  • Alternative: Include housekeeping protein controls and normalize signal

• Non-specific binding:

  • Cause: Cross-reactivity with other DEAD box proteins due to conserved motifs

  • Solution: Use antibodies targeting N-terminal regions where sequence divergence is greater

  • Alternative: Perform peptide competition assays to verify specificity

• Signal variability across applications:

  • Cause: Epitope accessibility differences between native and denatured states

  • Solution: Select antibodies validated for specific applications of interest

  • Alternative: Test multiple antibody clones targeting different epitopes

How are recent findings about DDX47 changing our understanding of cellular RNA metabolism?

Recent research has revealed several novel aspects of DDX47 function:

• R-loop stability regulation:

  • DDX47 maintains R-loop stability, with its depletion causing unscheduled R-loop accumulation

  • This disrupts normal RNA polymerase II function and creates transcription-replication conflicts

  • Biotinylated antibodies enable precise localization studies at sites of DNA:RNA hybrid formation

• Nucleolar stress responses:

  • DDX47 may serve as a sensor for nucleolar stress

  • Its relocalization correlates with altered rRNA processing

  • Biotinylated antibodies allow for live-cell tracking of DDX47 during stress responses

• Post-translational modification regulation:

  • DDX47 knockdown in model organisms leads to increased protein acetylation and crotonylation

  • This suggests a previously unrecognized role in epigenetic regulation

  • Biotinylated antibodies can be used in proximity ligation assays to identify interaction partners

• Immune system modulation:

  • As a member of the DDX family (intracellular nucleic acid recognition proteins)

  • DDX47 is involved in innate immune responses and regulates interferon production

  • Biotinylated antibodies enable sensitive detection of expression changes during immune challenges

What experimental systems are best suited for studying DDX47 function in disease models?

Several model systems have proven valuable for DDX47 research:

• Cell culture models:

  • Human cell lines: HeLa, HEK-293T, and SH-SY5Y show reliable DDX47 expression

  • Cancer cell lines with varying DDX47 expression can model disease-specific alterations

  • CRISPR/Cas9 gene editing allows precise functional studies

• Insect models:

  • Locusta migratoria has provided valuable insights into DDX47 function in tissue homeostasis

  • LmDDX47 knockdown causes midgut and gastric cecum atrophy

  • Affects regenerative and columnar cells in the midgut

  • Serves as a model for studying essential biological processes regulated by DDX47

• Clinical samples:

  • Sepsis and infection patient samples show diagnostic value of DDX47

  • Cancer tissue microarrays reveal prognostic significance in various cancer types

  • Biotinylated antibodies enhance detection sensitivity in limited clinical samples

• Novel methodological approaches:

  • Organoid systems to model tissue-specific DDX47 functions

  • Patient-derived xenografts for cancer-specific studies

  • High-throughput screening platforms to identify DDX47-targeting compounds

How can biotinylated DDX47 antibodies be integrated with advanced imaging techniques?

Combining biotinylated DDX47 antibodies with cutting-edge imaging enhances research capabilities:

• Super-resolution microscopy:

  • STORM/PALM techniques with streptavidin-fluorophore conjugates for nanoscale localization

  • Structured illumination microscopy (SIM) for detailed nucleolar organization

  • Expansion microscopy to physically magnify subcellular structures containing DDX47

• Multiplexed imaging approaches:

  • Sequential labeling with different streptavidin conjugates to visualize multiple targets

  • Mass cytometry (CyTOF) using metal-conjugated streptavidin for highly multiplexed analysis

  • Cyclic immunofluorescence to study DDX47 in the context of dozens of other proteins

• Live-cell imaging strategies:

  • Cell-permeable biotin ligands for pulse-chase experiments

  • SNAP-tag DDX47 fusions with biotin substrates

  • Time-lapse microscopy to track DDX47 dynamics during cell cycle or stress response

• Correlative light and electron microscopy (CLEM):

  • Biotinylated antibodies with gold-streptavidin for EM visualization

  • Precise ultrastructural localization of DDX47 at nucleolar organization centers

  • Integration with cryo-electron tomography for 3D structural context

By leveraging these advanced approaches, researchers can gain unprecedented insights into DDX47's localization, interactions, and functions in health and disease contexts.