DHX58 Antibody

What is DHX58 and why is it important in immunological research?

DHX58, also known as LGP2 (Laboratory of Genetics and Physiology 2), is a member of the RIG-I-like receptor (RLR) family that plays a crucial regulatory role in antiviral signaling pathways. Unlike other RLR family members, DHX58 lacks the CARD domain required for activating MAVS/IPS1-dependent signaling events, making it unable to initiate antiviral signaling independently . Its importance in immunological research stems from its dual regulatory functions - both negative and positive - in relation to RIGI and IFIH1/MDA5 signaling pathways .

DHX58 is particularly significant in studies of innate immune responses against various RNA viruses, some DNA viruses such as poxviruses and SARS-CoV-2, and even bacterial pathogens like Listeria monocytogenes . Its ability to bind both ssRNA and dsRNA, with higher affinity for dsRNA and preference for 5'-triphosphorylated RNA, makes it an intriguing target for understanding pathogen recognition mechanisms .

What are the main applications for DHX58 antibodies in research?

DHX58 antibodies are versatile tools employed across multiple experimental techniques in immunological and virological research. The primary applications include:

• Western Blotting (WB): Used to detect and quantify DHX58 protein expression levels, typically observing a band at approximately 77 kDa .

• Immunohistochemistry (IHC): Applied to visualize DHX58 distribution in paraffin-embedded tissue sections, offering insight into tissue-specific expression patterns .

• Immunoprecipitation (IP): Employed to isolate DHX58 protein complexes from tissue or cell lysates, facilitating the study of protein-protein interactions .

• Immunofluorescence (IF): Enables subcellular localization studies of DHX58 protein .

• Co-Immunoprecipitation (CoIP): Used to investigate interactions between DHX58 and other proteins in the antiviral signaling pathway .

• RNA Immunoprecipitation (RIP): Applied to study RNA-protein interactions involving DHX58, particularly relevant given its RNA-binding properties .

Most commercially available antibodies show reactivity with human, mouse, and rat samples, with some cross-reactivity reported for pig, monkey, and chicken samples .

How do I select the appropriate DHX58 antibody for my specific research needs?

Selecting the optimal DHX58 antibody requires careful consideration of multiple factors:

• Target species: Ensure the antibody has confirmed reactivity with your experimental model. Available antibodies show reactivity primarily with human, mouse, and rat samples, while some report cross-reactivity with pig, monkey, and chicken samples .

• Antibody type: Most DHX58 antibodies are rabbit polyclonal, though mouse monoclonal options are available for certain applications . Polyclonal antibodies typically offer higher sensitivity but potentially lower specificity than monoclonals.

• Target epitope: Consider which region of DHX58 you need to target. Available antibodies target various regions including:

  • N-terminal region (AA 41-69)

  • Central region (AA 200-250)

  • C-terminal regions (AA 389-678, AA 479-678)

  • Full-length protein (AA 1-678)

• Validated applications: Review the validated applications data and published literature citing the antibody for your intended technique. For example, if conducting IHC, prioritize antibodies with demonstrated success in this application .

• Immunogen information: Understanding the immunogen used to generate the antibody can help predict potential cross-reactivity and specificity issues .

For challenging applications or when studying poorly characterized samples, conducting preliminary validation experiments with multiple antibodies may be advisable.

What are the optimal conditions for Western blotting with DHX58 antibodies?

Successful Western blotting with DHX58 antibodies requires optimization of several key parameters:

Sample Preparation:

• For cellular samples: HEK-293 cells have been successfully used with DHX58 antibodies

• For tissue samples: Rat kidney, rat liver, and mouse liver tissues have shown positive detection

• Standard lysis buffers containing protease inhibitors are typically sufficient

Protocol Optimization:

• Protein loading: 20-50 μg of total protein per lane is generally recommended

• Antibody dilution: Start with manufacturer's recommended range (typically 1:200-1:1000 for DHX58 antibodies)

• Expected molecular weight: DHX58 appears at approximately 77 kDa

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Primary antibody incubation: Overnight at 4°C is typically optimal

• Secondary antibody: Anti-rabbit HRP conjugate (as most DHX58 antibodies are rabbit-derived)

Troubleshooting Tips:

• If background is high, increase blocking time or concentration, and optimize antibody dilution

• If signal is weak, consider longer exposure times, increased antibody concentration, or enhanced chemiluminescence substrates

• Always include positive control samples (HEK-293 cells or rat liver tissue) to verify antibody performance

Many DHX58 antibodies have been validated in multiple publications, providing confidence in their reliability for Western blot applications .

How should I optimize immunohistochemistry protocols for DHX58 detection in tissue samples?

Immunohistochemical detection of DHX58 requires careful attention to tissue preparation and staining conditions:

Tissue Processing and Antigen Retrieval:

• Fixation: 10% neutral buffered formalin is standard for most tissues

• Embedding: Paraffin embedding is commonly used

• Section thickness: 4-6 μm sections are optimal

• Antigen retrieval: Two recommended methods:

  • TE buffer pH 9.0 (preferred method)

  • Citrate buffer pH 6.0 (alternative method)

Staining Protocol:

• Deparaffinize and rehydrate sections following standard protocols

• Perform antigen retrieval (as above)

• Block endogenous peroxidase activity (3% H₂O₂, 10 minutes)

• Block non-specific binding (5-10% normal serum, 1 hour)

• Primary antibody dilution: Start with 1:50-1:500

• Incubation: Overnight at 4°C or 1-2 hours at room temperature

• Detection system: Compatible HRP-conjugated secondary antibody and DAB substrate

• Counterstain: Hematoxylin for nuclear visualization

Sample Selection and Controls:

• Positive tissue controls: Human breast cancer tissue and human nephroblastoma tissue have shown reliable DHX58 staining

• Always include negative controls (primary antibody omitted or isotype control)

Optimization Tips:

• Titrate antibody concentration based on staining intensity and background

• If background is high, increase blocking time and optimize antibody dilution

• If signal is weak, consider increasing antibody concentration or extending incubation time

The example below shows successful IHC staining of human breast cancer tissue using a DHX58 antibody at 1:10 dilution under 10x magnification .

What controls should be included when validating a new DHX58 antibody?

Thorough validation of DHX58 antibodies requires multiple types of controls to ensure specificity and reliability:

Positive Controls:

• Known positive cell lines: HEK-293 cells express detectable levels of DHX58

• Known positive tissues: Rat kidney, rat liver, and mouse liver tissues show reliable expression

• Recombinant DHX58 protein: Can be used as a standard for Western blotting

• Overexpression systems: Cells transfected with DHX58 expression constructs

Negative Controls:

• Knockout/knockdown validation: Samples from DHX58 knockout models or siRNA-treated cells to confirm antibody specificity

• Primary antibody omission: To identify non-specific binding of detection systems

• Isotype controls: To identify non-specific binding of the primary antibody

• Blocking peptide competition: Pre-incubation with immunizing peptide should abolish specific signal

Cross-reactivity Assessment:

• Test across multiple species if cross-species reactivity is claimed

• Verify specificity against related proteins within the RLR family (RIG-I, MDA5)

Multi-application Validation:

• Confirm consistent results across different techniques (WB, IHC, IF)

• Compare multiple antibodies targeting different epitopes within DHX58

This comprehensive validation approach has been employed in multiple publications utilizing DHX58 antibodies, with knockout/knockdown validation particularly valuable for confirming specificity .

How can DHX58 antibodies be used to investigate protein-protein interactions in antiviral signaling pathways?

DHX58 antibodies are valuable tools for elucidating the complex protein interaction network in antiviral signaling pathways:

Co-Immunoprecipitation (Co-IP) Approaches:

• Standard Co-IP: DHX58 antibodies can precipitate native protein complexes from cell or tissue lysates to identify interacting partners

• Reverse Co-IP: Antibodies against suspected interaction partners can be used to precipitate complexes, followed by DHX58 detection

• Crosslinking Co-IP: Chemical crosslinking prior to lysis can stabilize transient or weak interactions

Proximity-Based Approaches:

• Proximity Ligation Assay (PLA): Combines DHX58 antibodies with antibodies against suspected interacting proteins to visualize interactions in situ

• FRET/BRET analysis: When combined with fluorescent protein tagging approaches

Interaction Dynamics Studies:

• Sequential Co-IP: To identify multiprotein complexes containing DHX58

• Stimulus-dependent interaction studies: Examining how viral infection alters DHX58 interactions

• Domain-specific interaction mapping: Using antibodies targeting different regions of DHX58

Methodological Considerations:

• Buffer conditions: Use mild lysis conditions (NP-40 or Triton X-100 based buffers) to preserve protein-protein interactions

• Antibody amounts: 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate is recommended

• Controls: Include isotype controls and knockout/knockdown samples

Current research has identified interactions between DHX58 and other RLR family members (RIG-I and MDA5), as well as with viral RNA and components of the downstream signaling machinery like MAVS/IPS1 .

What methodological approaches can resolve contradictory findings in DHX58 functional studies?

The dual regulatory roles of DHX58 in antiviral signaling - both negative and positive - have led to seemingly contradictory findings in the literature. Several methodological approaches can help resolve these discrepancies:

Context-Dependent Functional Analysis:

• Viral specificity: Systematically test multiple virus types as DHX58's role may differ depending on the invading pathogen

• Cell type variation: Compare DHX58 function across multiple cell types relevant to viral infection (epithelial cells, immune cells, etc.)

• Temporal dynamics: Analyze DHX58 function at different time points post-infection

Mechanistic Dissection Approaches:

• Domain-specific mutants: Generate and study DHX58 mutants affecting specific functional domains

• Structure-function correlation: Use crystal structure data to inform functional studies

• Post-translational modification analysis: Investigate how modifications affect DHX58 activity

Integration of Multiple Readouts:

• Combine protein interaction studies (Co-IP) with functional readouts (interferon production)

• Correlate RNA binding activity with downstream signaling effects

• Employ both gain-of-function and loss-of-function approaches

The seemingly contradictory roles of DHX58 likely reflect its complex regulation of antiviral signaling, which may involve:

• Competition with RIG-I for viral RNA binding

• Direct binding to RIG-I, inhibiting its dimerization and interaction with MAVS/IPS1

• Competition with IKBKE for MAVS/IPS1 binding

• Facilitating RNA recognition by unwinding or stripping viral RNA of nucleoproteins

These mechanistic possibilities can be systematically evaluated using the approaches described above.

How can DHX58 antibodies be employed in RNA-protein interaction studies?

Given DHX58's RNA-binding properties, antibodies against this protein are valuable tools for investigating RNA-protein interactions:

RNA Immunoprecipitation (RIP) Approaches:

• Native RIP: Precipitate DHX58-RNA complexes under native conditions to identify associated RNAs

• Crosslinked RIP: UV or chemical crosslinking to stabilize transient RNA-protein interactions

• CLIP (Crosslinking and Immunoprecipitation): More stringent approach for mapping direct interaction sites

Methodological Considerations:

• RNase inhibition: Include RNase inhibitors throughout all procedures

• DNase treatment: To eliminate DNA contamination

• Controls: Include IgG control IPs and DHX58-depleted samples

• RNA analysis: qRT-PCR for known targets or RNA-seq for discovery approaches

Applications in Viral RNA Recognition Studies:

• Identifying viral RNA species recognized by DHX58

• Determining RNA structural features that enhance DHX58 binding

• Investigating competition between DHX58 and other RLRs for viral RNA

• Examining how RNA binding affects DHX58's protein interaction network

Current research indicates DHX58 can bind both ssRNA and dsRNA, with higher affinity for dsRNA. It shows preference for 5'-triphosphorylated RNA, although it can recognize RNA lacking a 5'-triphosphate . These binding preferences can be systematically investigated using RIP and CLIP approaches combined with specific DHX58 antibodies.

What strategies can address common issues with DHX58 antibody specificity?

Ensuring antibody specificity is critical for reliable DHX58 detection. Several strategies can address common specificity issues:

Antibody Validation Approaches:

• Genetic validation: Use samples from DHX58 knockout/knockdown models to confirm specificity

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

• Epitope mapping: Use truncated DHX58 constructs to confirm epitope specificity

• Multi-antibody comparison: Compare results with antibodies targeting different DHX58 epitopes

Technical Optimization for Improved Specificity:

• Titrate antibody concentration: Often, lower concentrations improve specificity

• Modify blocking conditions: Increase blocking reagent concentration or time

• Adjust incubation temperatures: Lower temperatures may reduce non-specific binding

• Use more stringent washing conditions: Increase wash buffer stringency or duration

Sample-Specific Considerations:

• Pre-adsorption: Pre-adsorb antibody with tissue/cell lysates from non-target species

• Modify lysis/extraction procedures: Adjust buffer composition to enhance target epitope accessibility

• Consider post-translational modifications: These may affect epitope recognition

Researchers should be particularly vigilant when working with DHX58 antibodies, as multiple proteins in the RLR family share sequence homology, potentially leading to cross-reactivity issues.

How should researchers interpret unexpected molecular weight variations in DHX58 Western blots?

The expected molecular weight of human DHX58 is approximately 77 kDa , but researchers may observe bands at different molecular weights. Here's how to interpret these variations:

Common Causes of Molecular Weight Variations:

Verification Strategies:

• Compare results across multiple DHX58 antibodies targeting different epitopes

• Perform knockout/knockdown validation experiments

• Use recombinant DHX58 protein standards of known molecular weight

• Analyze the same samples using alternative detection methods (mass spectrometry)

Experimental Considerations:

• Different species may show slight variations in DHX58 molecular weight

• Different gel systems and running conditions can affect apparent molecular weight

• Post-translational modifications are often cell type or stimulus dependent

What are the best practices for preserving DHX58 antibody activity during storage and handling?

Proper storage and handling of DHX58 antibodies is essential for maintaining their activity and reliability:

Storage Recommendations:

• Short-term storage (up to 2 weeks): Store at 4°C

• Long-term storage: Aliquot and store at -20°C

• Avoid repeated freeze-thaw cycles that can degrade antibody activity

• Some DHX58 antibodies are supplied in glycerol-containing buffers (typically 50% glycerol with PBS and 0.02% sodium azide)

Handling Best Practices:

• Aliquoting: Divide antibody into single-use aliquots upon receipt to avoid repeated freeze-thaw cycles

• Thawing: Thaw aliquots completely but gently (on ice or at 4°C)

• Centrifugation: Brief centrifugation after thawing to collect contents

• Temperature control: Keep antibodies on ice during experiment setup

• Contamination prevention: Use sterile techniques when handling antibody solutions

Reconstitution and Dilution:

• If lyophilized, reconstitute according to manufacturer's instructions

• Use high-quality, sterile diluents for preparing working solutions

• For working dilutions that will be stored, consider adding BSA (0.1-1%) as a stabilizer

• Record date of reconstitution and number of freeze-thaw cycles

Stability Assessment:

• Include positive controls to verify antibody activity over time

• Consider preparing activity/calibration curves for quantitative applications

• Be aware that antibody stability may be application-dependent (e.g., more stable for Western blotting than for immunoprecipitation)

Following these storage and handling guidelines will help ensure consistent results with DHX58 antibodies across experiments.

How can DHX58 antibodies contribute to COVID-19 and other viral pathogenesis research?

DHX58 has been implicated in the innate immune response to SARS-CoV-2 and other viruses, making DHX58 antibodies valuable tools for COVID-19 research:

Applications in SARS-CoV-2 Research:

• Expression profiling: Monitor DHX58 expression changes during SARS-CoV-2 infection using Western blotting or IHC

• Viral RNA recognition: Use RIP approaches to study DHX58 binding to SARS-CoV-2 RNA

• Signaling pathway analysis: Investigate DHX58's role in interferon responses to SARS-CoV-2

• Tissue distribution studies: Examine DHX58 expression in COVID-19 patient samples

Methodological Approaches:

• In vitro infection models: Study DHX58 responses in cell culture systems

• Ex vivo tissue analysis: Examine DHX58 expression in patient samples

• Animal models: Use DHX58 antibodies in animal models of viral infection

• Comparative virology: Compare DHX58 responses across different coronaviruses

Research Questions Addressable with DHX58 Antibodies:

• Does SARS-CoV-2 modulate DHX58 expression or localization?

• How does DHX58 contribute to the cytokine responses in COVID-19?

• Can DHX58 serve as a biomarker for disease severity or progression?

• Do genetic variants of DHX58 correlate with COVID-19 outcomes?

DHX58's involvement in innate immune responses to SARS-CoV-2 and other viruses makes it a promising target for understanding viral pathogenesis and potentially developing therapeutic strategies .

What considerations are important when studying post-translational modifications of DHX58?

Post-translational modifications (PTMs) can significantly impact DHX58 function. Studying these modifications requires specific methodological considerations:

Types of PTMs Relevant to DHX58:

• Phosphorylation: May regulate activity and protein interactions

• Ubiquitination: Potentially influences protein stability and signaling functions

• SUMOylation: May affect localization and regulatory functions

• ADP-ribosylation: Potentially involved in antiviral responses

Detection Approaches:

• Phospho-specific antibodies: If available for known DHX58 phosphorylation sites

• Modification-specific detection: General phospho-detection (e.g., ProQ Diamond) or ubiquitin detection methods

• Mass spectrometry: For comprehensive PTM mapping

• Migration shift analysis: PTMs often alter protein migration in gels

Methodological Considerations:

• Lysis buffer composition: Include phosphatase inhibitors (for phosphorylation) and deubiquitinase inhibitors (for ubiquitination)

• Stimulation conditions: Many PTMs are dynamically regulated following viral infection or interferon stimulation

• Subcellular fractionation: PTMs may affect DHX58 localization

• Mutation analysis: Generate phospho-mimetic or phospho-deficient mutants to study functional impacts

Challenges and Solutions:

• Low abundance of modified forms: May require enrichment strategies

• Dynamic regulation: Time-course experiments crucial for capturing transient modifications

• Site-specific effects: Different modification sites may have distinct functional consequences

• Stimulus specificity: PTM patterns may differ between virus types or other stimuli

Studying DHX58 PTMs can provide critical insights into its regulatory mechanisms and functional versatility in antiviral responses.

How can multi-omics approaches incorporating DHX58 antibodies enhance our understanding of antiviral immunity?

Integrating DHX58 antibody-based studies with multi-omics approaches can provide comprehensive insights into antiviral immunity networks:

Integrative Research Strategies:

• Proteomics + Antibody-Based Methods:

  • Use DHX58 immunoprecipitation coupled with mass spectrometry to identify interaction partners

  • Compare DHX58 interactomes across different viral infections

  • Correlate global proteome changes with DHX58 activation status

• Transcriptomics + DHX58 Studies:

  • Combine DHX58 ChIP-seq (if transcriptional roles are suspected) with RNA-seq

  • Correlate DHX58 activity with transcriptional responses to viral infection

  • Compare wild-type vs. DHX58-deficient transcriptional responses

• RNA-Protein Interactions:

  • Integrate RIP-seq data for DHX58 with transcriptome-wide analyses

  • Map DHX58 binding sites on viral and host RNAs

  • Correlate RNA binding with functional outcomes

Methodological Considerations:

• Sample preparation compatibility: Ensure methods are compatible across different omics platforms

• Temporal alignment: Coordinate timing of different analyses for meaningful integration

• Data integration approaches: Employ advanced bioinformatics for multi-omics data integration

• Validation strategies: Use antibody-based methods to validate key findings from omics studies

Potential Research Applications:

• Comprehensive mapping of DHX58-dependent antiviral response networks

• Identification of virus-specific DHX58 functions across different pathogen types

• Discovery of novel regulatory mechanisms controlling DHX58 activity

• Systems-level understanding of DHX58's dual regulatory roles

This multi-omics integration can help resolve the complex and sometimes contradictory functions of DHX58 in antiviral immunity by providing a more holistic view of its regulatory network.

What are the emerging technologies that may advance DHX58 antibody applications?

As antibody technologies continue to evolve, several emerging approaches hold promise for advancing DHX58 research:

• Single-domain antibodies and nanobodies: Smaller antibody formats that may offer improved tissue penetration and access to sterically hindered epitopes of DHX58, potentially revealing new aspects of its function.

• Antibody engineering for live-cell imaging: Development of intrabodies or cell-permeable antibody formats could enable real-time tracking of DHX58 dynamics during viral infection.

• Proximity labeling approaches: Combining DHX58 antibodies with enzymes like APEX2, BioID, or TurboID for proximity-dependent labeling can reveal the dynamic DHX58 interactome under different conditions.

• Single-cell antibody-based methods: Integration of DHX58 detection into single-cell proteomics workflows to understand cell-to-cell variation in antiviral responses.

• Super-resolution microscopy compatible antibodies: Development of DHX58 antibodies optimized for super-resolution techniques like STORM or PALM could reveal previously unobservable details of DHX58 localization and trafficking.

These technological advances, when applied to DHX58 research, promise to provide unprecedented insights into its complex regulatory functions in antiviral immunity and potentially reveal new therapeutic targets for viral diseases.