DDX4 Antibody, HRP conjugated

What is DDX4 and what are its primary biological functions?

DDX4, also known as DEAD-box helicase 4 or Vasa homolog (MVH), is an ATP-dependent RNA helicase that plays critical roles in multiple cellular processes. It primarily functions as a regulator of RNA secondary structure through RNA binding and is essential for several biological processes:

• Germline development: DDX4 is required during spermatogenesis and is essential for germline integrity .

• RNA processing: It supports various RNA processes including splicing, transport, and stability within germ cells .

• Transposon repression: DDX4 represses transposable elements and prevents their mobilization, which is essential for maintaining genomic integrity in germline cells .

• piRNA metabolism: It mediates the repression of transposable elements during meiosis by forming complexes with piRNAs and Piwi proteins, governing methylation and subsequent repression of transposons .

• Antiviral immunity: Recent research has revealed that DDX4 acts as a positive regulatory molecule in Type-I interferon (IFN-I)-mediated antiviral activity, creating a positive feedback loop that amplifies antiviral responses .

• Cancer progression: In certain cancers, DDX4 contributes to increased cell motility and drug resistance, particularly in small cell lung cancer (SCLC) where it enhances cisplatin resistance .

DDX4's diverse functions make it an important target for studies in reproductive biology, cancer research, and immunology.

What experimental applications are suitable for DDX4 HRP-conjugated antibodies?

DDX4 HRP-conjugated antibodies can be used in multiple experimental applications with specific optimization parameters:

For Western blotting applications, researchers should expect to detect DDX4 at approximately 80 kDa molecular weight . The HRP conjugation eliminates the need for secondary antibody incubation, streamlining the experimental workflow and potentially reducing background.

When using DDX4 antibodies for immunofluorescence, frozen tissue sections generally yield better results than paraffin-embedded sections, particularly for germline tissues. The 1:200 dilution recommended for immunofluorescence applications should be optimized based on the specific tissue type and fixation method .

What are the technical advantages of using HRP-conjugated DDX4 antibodies compared to unconjugated versions?

HRP (Horseradish Peroxidase) conjugation provides several significant advantages for DDX4 detection in research settings:

• Simplified workflow: Direct conjugation eliminates the need for secondary antibody incubation, reducing experimental time by approximately 1-2 hours and minimizing potential sources of variability.

• Reduced background: The absence of secondary antibodies decreases non-specific binding, particularly important when studying tissues with high endogenous biotin or immunoglobulin content.

• Enhanced sensitivity: Direct enzyme linkage allows for more efficient signal generation upon substrate addition, potentially improving detection of low-abundance DDX4 expression in non-germline tissues.

• Versatile applications: HRP-conjugated antibodies can be used across multiple detection platforms, including immunohistochemistry, Western blotting, and ELISA.

• Quantitative analysis: The signal produced is proportional to antibody binding, allowing for better quantification of DDX4 expression levels across different experimental conditions.

When using HRP-conjugated DDX4 antibodies, researchers should consider:

• Optimizing enzyme substrate exposure time to prevent oversaturation of signal

• Implementing proper blocking steps to minimize non-specific binding

• Considering enzyme stability during long-term storage to maintain consistent performance

How should researchers optimize sample preparation for DDX4 detection in different tissue types?

Sample preparation is critical for successful DDX4 detection, with requirements varying by tissue type and experimental goal:

For germline tissues (primary DDX4 expression sites):

• Fix tissues in 4% paraformaldehyde for 4-6 hours at 4°C to preserve protein structure

• For Western blotting, supplement lysis buffers with RNase inhibitors to prevent degradation of RNA-protein complexes

• When performing immunofluorescence, include a permeabilization step (0.2% Triton X-100, 10 minutes) to access intracellular DDX4

For cancer tissues (aberrant DDX4 expression):

• Consider shorter fixation times (2-4 hours) to prevent antigen masking

• Implement antigen retrieval using citrate buffer (pH 6.0) for 15-20 minutes

• Include positive controls (germline tissues) alongside cancer samples to validate detection methods

For tissues under viral infection:

• Collect samples at multiple time points post-infection to capture dynamic changes in DDX4 expression

• Consider dual staining with viral markers to correlate DDX4 expression with infection progression

Regardless of tissue type, researchers should:

• Validate antibody specificity using appropriate positive and negative controls

• Optimize antibody concentration through titration experiments

• Consider signal amplification methods for detecting low-abundance expression

How can researchers interpret DDX4 expression patterns across different experimental contexts?

Interpreting DDX4 expression requires understanding its context-dependent roles and expression patterns:

When analyzing DDX4 expression, researchers should:

• Consider subcellular localization (typically cytoplasmic in germline cells)

• Quantify expression levels relative to appropriate controls

• Correlate expression with functional outcomes (e.g., viral replication efficiency, drug sensitivity)

• Examine co-expression with pathway-related proteins (e.g., interferon signaling components, USP7, SOCS1)

The relevance of DDX4 expression varies significantly between experimental contexts, requiring careful interpretation based on the specific research question.

How can researchers design experiments to investigate DDX4's role in antiviral immune responses?

Recent discoveries about DDX4's involvement in antiviral immunity provide new research opportunities. Based on findings that DDX4 enhances Type-I interferon (IFN-I) signaling, researchers can design comprehensive experimental approaches:

Experimental design for DDX4-antiviral studies:

• Gene modulation approaches:

  • Generate DDX4 knockout cell lines using CRISPR-Cas9 to assess viral susceptibility

  • Create DDX4 overexpression systems to evaluate enhanced antiviral protection

  • Develop inducible expression systems to study temporal effects of DDX4 upregulation

• Viral infection models:

  • Compare multiple virus types (RNA viruses like VSV, SeV, H1N1 and DNA viruses like HSV) to assess broad-spectrum effects

  • Monitor viral replication kinetics at 12, 24, and 36-hour timepoints post-infection

  • Quantify viral proteins, viral RNA, and viral titers in DDX4-modulated systems

• Signaling pathway analysis:

  • Examine JAK-STAT pathway activation through phosphorylation status

  • Monitor SOCS1 protein levels and stability in relation to DDX4 expression

  • Investigate the DDX4-USP7-SOCS1 regulatory axis using co-immunoprecipitation

• Ubiquitination studies:

  • Analyze SOCS1 ubiquitination levels in DDX4 knockout and overexpression systems

  • Focus on K48-linked ubiquitination at the SOCS1-K119 residue

  • Employ ubiquitin mutants to confirm linkage specificity

Key findings from recent research:

• DDX4 knockout macrophages (RAW264.7 cells) showed higher viral protein and RNA levels compared to wild-type cells when infected with VSV or H1N1

• Overexpression of DDX4 decreased viral replication across multiple cell lines (RAW264.7, A549, 2fTGH, HEK293T)

• DDX4 forms a positive feedback loop with IFN-I, as interferon upregulates DDX4, which then enhances interferon signaling

• The antiviral mechanism involves DDX4 binding to USP7, disrupting USP7-SOCS1 interaction, leading to SOCS1 degradation and enhanced interferon activity

These experimental approaches can reveal novel insights into unconventional roles of germline factors in immunity.

What are the optimal experimental conditions for detecting DDX4 in tissues with low expression levels?

Detecting DDX4 in non-germline tissues where expression may be low presents significant technical challenges. Here are optimized approaches for enhanced sensitivity:

Sample preparation optimization:

• Tissue fixation and processing:

  • Use shorter fixation times (2-4 hours) with 4% paraformaldehyde at 4°C

  • Process tissues rapidly to minimize protein degradation

  • For frozen sections, use optimal cutting temperature (OCT) compound and maintain consistent 8-10 μm section thickness

• Antigen retrieval strategies:

  • Implement heat-induced epitope retrieval using citrate buffer (pH 6.0) for 15-20 minutes

  • Test multiple retrieval methods in parallel to determine optimal conditions

  • Consider enzymatic retrieval as an alternative for certain tissue types

• Signal amplification methods:

  • Employ tyramide signal amplification (TSA) to enhance HRP signal by 10-50 fold

  • Consider biotin-free amplification systems to reduce background in endogenous biotin-rich tissues

  • Use highly sensitive chemiluminescent substrates for Western blotting applications

Protocol for enhanced immunofluorescence detection:

Validation approaches:

• Include known positive controls (germline tissues) alongside test samples

• Perform parallel detection with multiple DDX4 antibodies targeting different epitopes

• Confirm specificity through genetic approaches (siRNA knockdown or CRISPR knockout)

• Use orthogonal methods (RT-qPCR, Western blotting) to validate protein detection

These optimized conditions can significantly improve detection sensitivity for DDX4 in cancer cells or tissues responding to viral infection, where expression levels may be substantially lower than in germline tissues.

How can researchers investigate DDX4's role in cancer chemoresistance mechanisms?

DDX4's emerging role in cancer progression and chemoresistance, particularly in small cell lung cancer (SCLC), presents important research opportunities. Based on recent findings , researchers can implement comprehensive experimental approaches:

Experimental design for studying DDX4-mediated chemoresistance:

• Cell line models:

  • Establish DDX4 knockout and overexpression in cancer cell lines (e.g., H69AR and SHP77 for SCLC studies)

  • Develop cisplatin-resistant cell lines to examine DDX4 expression changes during resistance acquisition

  • Create isogenic cell line pairs differing only in DDX4 expression for direct comparison

• Drug sensitivity assays:

  • Perform dose-response curves with cisplatin and other chemotherapeutics

  • Calculate IC50 values in DDX4-modulated systems

  • Conduct time-course studies to assess resistance development kinetics

• Molecular mechanism investigations:

  • Perform proteomic analysis of DDX4-expressing versus non-expressing cells

  • Focus on DNA repair and immune/inflammatory response pathways

  • Investigate changes in mRNA translation efficiency

• In vivo xenograft models:

  • Compare tumor growth kinetics of DDX4-expressing versus DDX4-depleted cancer cells

  • Assess response to cisplatin treatment in established tumors

  • Perform ex vivo analysis of harvested tumors for molecular markers

Key findings and their implications:

• DDX4 expression increases drug resistance, motility, and mRNA translation in SCLC cells

• Proteomic analysis reveals DDX4 upregulates proteins related to DNA repair and immune/inflammatory response pathways

• DDX4 depletion compromises tumor development in mouse models, while overexpression enhances tumor growth even after cisplatin treatment

• Higher DDX4 expression in SCLC patients correlates with decreased survival, suggesting clinical relevance

Researchers should consider studying:

• How DDX4's RNA helicase activity contributes to chemoresistance

• The relationship between DDX4's germline functions and its role in cancer

• Potential for DDX4 as a biomarker for treatment response prediction

• Development of strategies to target DDX4 or its downstream pathways therapeutically

How can researchers study the protein-protein interactions of DDX4 with components of the USP7/SOCS1 pathway?

Investigating DDX4's interactions with the USP7/SOCS1 pathway requires sophisticated methods to capture both stable and transient protein complexes. Recent research has revealed that DDX4 binds USP7, disrupting the USP7-SOCS1 interaction and affecting antiviral responses .

Recommended experimental approaches:

• Co-immunoprecipitation (Co-IP) studies:

  • Perform reciprocal Co-IP experiments using DDX4-HRP antibodies to pull down complexes and detect USP7

  • Use HA-USP7 and Myc-DDX4 tagged constructs for exogenous expression systems

  • Include appropriate controls (IgG control, input lysate) to verify specificity

  • Optimize lysis conditions to preserve intact protein complexes

• Proximity-based interaction assays:

  • Implement proximity ligation assay (PLA) to visualize DDX4-USP7 interactions in situ

  • Consider BioID or APEX2 proximity labeling with DDX4 as the bait to identify novel interaction partners

  • Use FRET-based approaches for measuring interaction dynamics in living cells

• Domain mapping studies:

  • Create truncation mutants of DDX4 to identify regions required for USP7 binding

  • Perform peptide array analysis to define specific interaction motifs

  • Use site-directed mutagenesis to confirm critical residues for protein-protein interactions

• Functional validation:

  • Assess how mutations affecting DDX4-USP7 binding impact SOCS1 stability

  • Measure changes in interferon signaling when the interaction is disrupted

  • Evaluate antiviral activity in cells expressing interaction-deficient mutants

Protocol for analyzing DDX4-mediated effects on SOCS1 ubiquitination:

Key research findings:

• DDX4 binding to USP7 disrupts the USP7-SOCS1 interaction

• This disruption leads to increased SOCS1 ubiquitination and subsequent degradation

• The mechanism specifically affects K48-linked ubiquitination at the K119 residue of SOCS1

• DDX4 cannot promote SOCS1 ubiquitination in USP7-deficient cells, confirming the USP7-dependent mechanism

Understanding these molecular interactions provides insight into how a germline factor unexpectedly regulates antiviral immunity through protein complex reorganization.

How can researchers integrate DDX4 expression data with functional outcomes in translational research?

Translating DDX4 research findings into clinically relevant contexts requires integrating expression data with functional outcomes across multiple research platforms:

Strategies for integrative analysis:

• Patient sample correlation studies:

  • Analyze DDX4 expression in patient-derived samples using immunohistochemistry

  • Correlate expression levels with clinical parameters (survival, treatment response)

  • Stratify patients based on DDX4 expression to identify potential prognostic subgroups

• Multi-omics integration:

  • Combine proteomics, transcriptomics, and functional assays to create comprehensive datasets

  • Identify pathway enrichment patterns associated with DDX4 expression

  • Focus on DNA repair and immune/inflammatory response pathways highlighted in recent research

• Therapeutic response prediction:

  • Develop predictive models based on DDX4 expression and associated pathways

  • Test predictive power using retrospective analysis of treatment outcomes

  • Design prospective validation studies in specific cancer types like SCLC

• Experimental validation pipeline:

  • Start with in vitro cellular models to establish mechanism

  • Proceed to xenograft models to validate in vivo relevance

  • Correlate findings with patient data to confirm clinical significance

Applications in cancer research:

• DDX4 expression correlates with decreased survival in SCLC patients, suggesting potential as a prognostic biomarker

• Proteomic changes induced by DDX4 include upregulation of proteins involved in DNA repair and immune/inflammatory responses, which may explain chemoresistance mechanisms

• DDX4 depletion compromises tumor development while its overexpression enhances tumor growth even after cisplatin treatment in experimental models

Applications in immunology research:

• DDX4's role in enhancing IFN-I signaling suggests potential applications in modulating antiviral responses

• The DDX4-USP7-SOCS1 regulatory axis represents a novel therapeutic target for enhancing interferon efficacy

• Understanding this pathway could lead to improved strategies for treating viral infections

When designing translational research projects involving DDX4:

• Consider both direct and indirect effects of DDX4 on cellular functions

• Examine potential tissue specificity of mechanisms

• Develop robust biomarker assays that can be standardized across clinical laboratories

• Investigate combination approaches that target DDX4-dependent pathways

This integrative approach enables researchers to move beyond descriptive studies to develop mechanistic insights with therapeutic potential.