DDX47 Antibody, FITC conjugated

What is DDX47 and why is it an important research target?

DDX47 is a DEAD box protein characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD). It functions as an ATP-dependent RNA helicase required for efficient ribosome biogenesis and is involved in mRNA splicing and apoptosis . Recent research has identified DDX47 as an R-loop resolvase that prevents R-loop-associated DNA damage . Its role in preventing harmful R-loops and transcription-replication conflicts makes it a valuable target for studying genomic stability and cancer biology.

What types of DDX47 antibodies are available for research applications?

Several types of DDX47 antibodies are available for research applications:

How do I properly store and handle DDX47 antibodies to maintain activity?

For optimal antibody performance:

• Store unconjugated antibodies at -20°C upon delivery, making aliquots to avoid freeze-thaw cycles .

• For FITC or fluorophore-conjugated antibodies, store in light-protected vials or covered with aluminum foil to prevent photobleaching .

• Short-term storage (less than 1 week) can be at 4°C .

• For long-term storage (up to 1 year), 50% glycerol is recommended when storing at -20°C .

• Conjugated antibodies remain stable for at least 12 months at 4°C, but longer storage (24 months) requires dilution with up to 50% glycerol and storage at -20°C to -80°C .

• Remember that repeated freezing and thawing will compromise enzyme activity and antibody binding capacity .

What are the recommended dilutions for different applications of DDX47 antibodies?

The optimal antibody dilution varies by application method:

Always perform a titration experiment to determine the optimal antibody concentration for your specific sample type and experimental conditions .

How can I confirm the specificity of DDX47 antibody in my experimental system?

To validate antibody specificity:

• Positive controls: Use cell lines known to express DDX47 such as HeLa, HepG2, Jurkat, SH-SY5Y, RT4, U-251 MG cells .

• Western blot analysis: Confirm a single band at the expected molecular weight (~50 kDa) .

• siRNA knockdown: Compare antibody signal between control and DDX47-depleted samples to confirm specificity .

• Immunofluorescence pattern: DDX47 should show a specific subcellular localization pattern consistent with its functions in the nucleus/nucleolus .

• Blocking peptide: Pre-incubate the antibody with the immunizing peptide to confirm that the signal is specific .

What methodologies can be used to study DDX47's role in R-loop resolution?

Recent research has identified DDX47 as an R-loop resolvase. To study this function:

• DNA-RNA hybrid immunoprecipitation (DRIP): Use the S9.6 antibody to detect R-loops before and after DDX47 depletion .

• Proximity ligation assay (PLA): Detect the association between DDX47 and DNA-RNA hybrids using anti-DDX47 and S9.6 antibodies .

• Chromatin immunoprecipitation (ChIP): Determine recruitment of DDX47 to chromatin regions .

• Immunofluorescence with S9.6 antibody: Visualize R-loop accumulation following DDX47 depletion .

• RNase H treatment: Confirm R-loop specificity by treating samples with RNase H, which specifically degrades RNA in DNA-RNA hybrids .

How can fluorescently-conjugated antibodies enhance DDX47 research applications?

While the search results specifically mention FITC-conjugated antibodies for DDX4 rather than DDX47, the principles of fluorescent conjugation apply similarly:

• Multicolor flow cytometry: FITC-conjugated antibodies (excitation/emission: 488nm/525nm) can be combined with other fluorophores for multiparameter analysis .

• Live cell imaging: Direct conjugation eliminates the need for secondary antibodies, reducing background and enabling real-time tracking .

• Co-localization studies: FITC-conjugated DDX47 antibodies can be paired with antibodies against interacting proteins labeled with different fluorophores (like Texas Red or Cy5) .

• FACS sorting: Fluorophore-conjugated antibodies enable isolation of cell populations based on DDX47 expression levels .

• Quantitative analysis: Direct fluorophore conjugation allows more accurate quantification of protein expression levels compared to indirect methods .

What techniques can be used to investigate DDX47's interactions with other proteins in the RNA processing machinery?

To study DDX47's protein interactions:

• Co-immunoprecipitation (Co-IP): Use anti-DDX47 antibodies to pull down DDX47 and its binding partners for Western blot analysis .

• Proximity Ligation Assay (PLA): Detect protein-protein interactions in situ with <100nm resolution .

• Bimolecular Fluorescence Complementation (BiFC): Directly visualize protein interactions in living cells.

• ChIP-seq combined with RNA-seq: Identify genomic regions where DDX47 is recruited and correlate with transcriptional output.

• Mass spectrometry after IP: Identify novel interaction partners of DDX47 in different cellular contexts .

How can I investigate the role of DDX47 in transcription-replication conflicts (TRCs)?

DDX47 has been implicated in preventing harmful transcription-replication conflicts:

• Proximity Ligation Assay (PLA): Use antibodies against RNAPI and PCNA to detect increased TRCs upon DDX47 depletion .

• EdU labeling: Assess DNA replication patterns in the presence and absence of DDX47.

• γH2AX immunostaining: Quantify DNA damage foci formation when DDX47 is depleted .

• DNA combing: Directly visualize replication fork progression and stalling.

• Chromatin fractionation: Compare protein recruitment to chromatin in control versus DDX47-depleted cells.

What are common challenges when using DDX47 antibodies and how can they be addressed?

How can I accurately quantify DDX47 expression levels in different cell types or tissue samples?

For accurate DDX47 quantification:

• Western blot with normalization: Normalize DDX47 signal to housekeeping proteins (β-actin, GAPDH) and use standard curves with recombinant DDX47 protein .

• qRT-PCR: Compare mRNA levels with protein levels to identify potential post-transcriptional regulation.

• Flow cytometry: Quantify DDX47 expression at the single-cell level using properly titrated antibodies .

• Enzyme-linked immunosorbent assay (ELISA): Develop a sandwich ELISA using different DDX47 antibodies recognizing distinct epitopes .

• Immunohistochemistry with digital image analysis: Use software tools to quantify staining intensity in tissue sections .

How should I interpret DDX47 localization patterns in relation to its various cellular functions?

DDX47 exhibits distinct localization patterns related to its functions:

• Nucleolar localization: Associated with its role in ribosome biogenesis .

• Nucleoplasmic localization: Related to its function in mRNA splicing and R-loop resolution .

• Cytoplasmic relocalization: May occur during cellular stress or apoptosis induction .

When interpreting localization data:

• Compare with markers for specific cellular compartments (nucleolin for nucleoli, SC35 for splicing speckles)

• Consider cell cycle stage, as localization may vary throughout the cell cycle

• Assess changes in response to transcriptional inhibition or replication stress, which can alter DDX47 distribution

• Verify patterns with multiple antibodies recognizing different epitopes to rule out artifacts

How can DDX47 antibodies be used to investigate its role in cancer and disease pathogenesis?

DDX47's involvement in fundamental cellular processes suggests important roles in disease:

• Tissue microarray analysis: Compare DDX47 expression across normal and tumor samples from different tissues .

• Patient-derived xenograft models: Track DDX47 expression during disease progression using immunohistochemistry.

• Correlation with clinical parameters: Analyze DDX47 expression in relation to patient survival, treatment response, and disease progression.

• Co-expression with cancer biomarkers: Determine how DDX47 expression correlates with established diagnostic or prognostic markers.

• Association with genomic instability: Investigate whether DDX47 dysfunction contributes to R-loop accumulation and genomic instability in cancer cells .

What are the emerging techniques for studying DDX47's enzymatic activity and how can antibodies facilitate these approaches?

To study DDX47's helicase activity:

• In vitro helicase assays: Assess purified DDX47's ability to unwind DNA-RNA hybrids using fluorescently labeled substrates.

• ATP hydrolysis assays: Measure ATPase activity in the presence of different RNA substrates.

• Single-molecule approaches: Directly visualize DDX47's action on individual nucleic acid molecules.

• Structure-function analysis: Use antibodies recognizing specific domains to determine which regions are required for different activities.

• Drug screening: Identify small molecules that modulate DDX47's enzymatic functions using antibody-based readouts.

How can advanced imaging techniques enhance our understanding of DDX47 dynamics in living cells?

Cutting-edge imaging approaches for DDX47 research:

• Super-resolution microscopy: Techniques like STORM, PALM, or SIM can reveal DDX47 distribution at nanoscale resolution, beyond the diffraction limit.

• Live-cell imaging: Using fluorescently-tagged DDX47 or antibody fragments to track its dynamics in response to cellular stress.

• FRAP (Fluorescence Recovery After Photobleaching): Measure mobility and binding kinetics of DDX47 in different cellular compartments.

• FRET (Förster Resonance Energy Transfer): Detect interactions between DDX47 and partner proteins with nanometer precision.

• Correlative light and electron microscopy (CLEM): Combine fluorescence imaging of DDX47 with ultrastructural context from electron microscopy.