DHX34 Antibody

What is DHX34 and why is it significant in molecular biology research?

DHX34 is a member of the DExD/H-box RNA helicase family that functions as a critical regulator in nonsense-mediated decay (NMD) and pre-mRNA splicing processes. It contains highly conserved DExH/D helicase core consisting of two recombinase A (RecA)-like helicase domains with conserved sequence motifs (I–VI) responsible for ATP binding, ATPase activity, and RNA binding functions . The significance of DHX34 has been highlighted by its involvement in cancer development, particularly in myelodysplasia (MDS) and acute myeloid leukemia (AML), making it an important target for both basic and translational research .

What types of DHX34 antibodies are available for research applications?

Current research primarily utilizes rabbit polyclonal antibodies targeting different regions of the DHX34 protein:

Most commercially available DHX34 antibodies have been validated primarily for Western blotting applications, with recommended working concentrations of approximately 1 μg/mL .

What are the optimal storage and handling conditions for DHX34 antibodies?

For short-term use (up to 1 week), DHX34 antibodies can be stored at 2-8°C. For long-term storage, aliquoting and storing at -20°C is recommended to prevent freeze-thaw cycles that may compromise antibody integrity. Most commercial DHX34 antibodies are supplied in PBS buffer with 0.09% (w/v) sodium azide and 2% sucrose . When working with these antibodies, it's advisable to equilibrate to room temperature before opening vials to prevent condensation which could affect antibody stability.

What are the validated applications for DHX34 antibodies in current research?

DHX34 antibodies have been validated primarily for the following applications:

• Western Blotting (WB): The most widely validated application, typically using 1 μg/mL concentration for optimal results .

• Immunoprecipitation (IP): Successfully employed to study protein-protein interactions of DHX34 with partners like UPF1, SMG1, SMG7, SMG9, SMG6, and UPF3a .

• Cross-Linking Immunoprecipitation (CLIP): Used to map DHX34 endogenous binding sites, revealing preferential association with pre-mRNAs at exon-intron boundaries .

• Immunohistochemical staining (IHC): Applied to analyze DHX34 expression in tissue samples, particularly in cancer research .

• mRNA capture assays: Used in conjunction with DHX34 antibodies to study direct RNA binding properties .

How can I optimize Western blot protocols for DHX34 detection?

For optimal Western blot detection of DHX34:

• Sample preparation: Use RIPA or NP-40 based lysis buffers with protease inhibitors.

• Protein loading: Load 20-50 μg of total protein per lane.

• Gel selection: Use 8-10% SDS-PAGE gels due to DHX34's relatively large size.

• Transfer conditions: Transfer to PVDF membranes at 100V for 90 minutes in standard Tris-glycine buffer.

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

• Primary antibody: Dilute DHX34 antibody to 1 μg/mL in blocking buffer and incubate overnight at 4°C .

• Detection: Standard HRP-conjugated secondary antibodies with ECL detection systems are suitable for visualization.

• Controls: Include positive control samples from cell lines with known DHX34 expression (e.g., HEK293T cells) .

What are the key considerations for immunoprecipitation experiments using DHX34 antibodies?

When performing immunoprecipitation with DHX34 antibodies:

• RNase treatment: Consider whether RNA-dependent interactions are of interest. For studying direct protein-protein interactions, include RNase treatment to eliminate RNA-mediated associations .

• Lysis conditions: Use gentle lysis buffers (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate) with protease inhibitors.

• Pre-clearing: Pre-clear lysates with appropriate control IgG to reduce non-specific binding.

• Antibody amount: Use 2-5 μg of DHX34 antibody per 500 μg of total protein.

• Washing stringency: Adjust washing stringency based on interaction strength; start with moderate stringency (150-300 mM NaCl).

• Elution method: Consider native elution with peptide competition when studying complex integrity.

• Verification: Always verify IP efficiency by Western blotting a small fraction of the immunoprecipitated material .

How can DHX34 antibodies be employed to study its dual role in NMD and pre-mRNA splicing?

To investigate DHX34's dual functionality:

• Differential protein complex analysis: Use DHX34 antibodies for co-immunoprecipitation followed by mass spectrometry to identify differential protein interactions under conditions that favor NMD versus splicing. Compare results with immunoprecipitations of known NMD factors (UPF1) and splicing regulators .

• Sequential ChIP/CLIP approaches: Perform sequential chromatin immunoprecipitation or CLIP using DHX34 antibodies followed by antibodies against splicing or NMD factors to identify sites of co-regulation.

• Functional rescue experiments: In DHX34-depleted cells, perform rescue experiments with wild-type and mutant DHX34 (with compromised splicing or NMD functions), then use DHX34 antibodies to confirm expression levels and localization patterns .

• RNA-seq analysis coupled with DHX34 occupancy: Combine RNA-seq from DHX34-depleted cells with DHX34 CLIP-seq data to correlate direct binding events with functional outcomes in splicing and NMD .

What approaches can be used to study DHX34 in cancer, particularly in myeloid malignancies?

For investigating DHX34 in cancer contexts:

• Comparative expression analysis: Use DHX34 antibodies for immunohistochemistry and Western blotting to compare expression levels between normal and malignant tissues, particularly in MDS/AML samples .

• Single-cell analysis: Apply DHX34 antibodies in single-cell Western blot or mass cytometry to profile heterogeneity in DHX34 expression within tumor populations.

• Functional studies in hematopoietic cells: Use DHX34 antibodies to monitor protein levels following knockdown or overexpression experiments in hematopoietic stem/progenitor cells (HSPCs) to correlate with differentiation markers (CD14, CD71) by flow cytometry .

• Mutation-specific antibody approaches: For studying DHX34 variants found in familial MDS/AML, consider developing mutation-specific antibodies or epitope-tagging approaches to distinguish wild-type from mutant proteins.

• Prognostic correlation studies: Utilize DHX34 antibodies for tissue microarray analysis to correlate expression levels with patient outcomes, immunotherapy response, and chemotherapy sensitivity .

How can I investigate the ATP-dependent helicase activity of DHX34 using immunological approaches?

To study DHX34's helicase activity:

• Purification of active DHX34: Use DHX34 antibodies for immunoaffinity purification of the native protein from cellular extracts while preserving its enzymatic activity.

• ATPase activity assays: Immunoprecipitate DHX34 using specific antibodies and measure ATP hydrolysis in the immunoprecipitated material.

• Mutant analysis: Compare wild-type DHX34 with K191S (defective in ATP binding) and D279A (defective in ATP hydrolysis) mutants using DHX34 antibodies to assess protein levels, then correlate with functional outcomes .

• RNA binding studies: Combine UV-crosslinking with DHX34 immunoprecipitation to study how ATP binding/hydrolysis affects RNA interactions. The D279A mutation, which abrogates ATP hydrolysis, shows increased mRNA binding, suggesting ATP hydrolysis is required for DHX34 release from RNA .

• Conformational antibodies: Consider developing conformation-specific antibodies that recognize ATP-bound versus ATP-free states of DHX34.

How can I validate DHX34 antibody specificity for my specific research application?

To ensure DHX34 antibody specificity:

• Knockout/knockdown controls: Validate antibody specificity using DHX34 knockout cell lines or siRNA-mediated knockdown. For example, RNA-seq experiments in HeLa cells depleted of DHX34 showed specific changes in alternative splicing patterns that could be detected by RT-PCR, providing a functional validation system .

• Peptide competition assays: Perform pre-adsorption of the antibody with the immunizing peptide (e.g., VPGRLFPITVVYQPQEAEPTTSKSEKLDPRPFLRVLESIDHKYPPEERGD for middle region antibodies) to confirm signal specificity .

• Multiple antibody validation: Use different antibodies targeting distinct epitopes of DHX34 to confirm consistent results.

• Recombinant protein controls: Use purified recombinant DHX34 as a positive control for Western blotting applications.

• Cross-reactivity testing: Test reactivity against closely related DExD/H-box family members to ensure specificity.

What are common pitfalls in DHX34 detection and how can they be overcome?

Common challenges and solutions include:

• Low signal intensity:

  • Increase antibody concentration (up to 2-5 μg/mL)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use signal enhancement systems (tyramide signal amplification)

  • Optimize protein extraction with specialized buffers for nuclear proteins

• High background:

  • Use fresher antibody aliquots to avoid aggregation

  • Increase blocking time and washing steps

  • Consider alternative blocking reagents (BSA instead of milk)

  • Titrate antibody to find optimal concentration

• Multiple bands in Western blots:

  • These could represent alternatively spliced variants (especially relevant for DHX34 which undergoes AS-NMD in AML)

  • Use cell lines with known DHX34 splice variants as controls

  • Consider using isoform-specific antibodies if available

• Inconsistent immunoprecipitation:

  • Pre-clear lysates thoroughly

  • Increase antibody amount or incubation time

  • Use protein A/G mix beads instead of either alone

  • Consider crosslinking antibody to beads to prevent co-elution

How should I analyze and interpret DHX34 expression data across different cancer types?

For accurate analysis of DHX34 expression data:

• Standardization approaches: Use consistent loading controls and quantification methods when comparing DHX34 expression across samples. For Western blot analysis, consider normalizing to multiple housekeeping proteins.

• Cancer-specific considerations: DHX34 expression varies significantly across cancer types. For example, DHX34 is highly expressed in liver hepatocellular carcinoma (LIHC) compared to normal tissues, but expression patterns may differ in other cancers .

• Correlation with clinical parameters: When analyzing DHX34 expression in clinical samples, correlate with:

  • Tumor stage and grade

  • Mutational burden (TMB) and microsatellite instability (MSI)

  • Patient survival data

  • Immunotherapy response metrics

• Single-cell analysis: In heterogeneous samples, consider that DHX34 expression may vary between cell types. In liver cancer, for instance, monocytes or macrophages exhibit higher expression levels of DHX34 compared to other cell types .

• Functional relevance thresholds: Determine expression level thresholds that correlate with functional outcomes (e.g., splicing changes, NMD efficiency) rather than simply classifying as "high" or "low" expression.

How can DHX34 antibodies contribute to studying its potential as a biomarker and therapeutic target?

DHX34's emerging role as a biomarker can be investigated using:

• Multiplex immunoassays: Develop DHX34 antibody-based multiplex assays that simultaneously detect DHX34 and other potential cancer biomarkers for improved diagnostic accuracy.

• Liquid biopsy applications: Explore the potential of detecting DHX34 protein in exosomes or circulating tumor cells using highly specific antibodies.

• Companion diagnostic development: Utilize DHX34 antibodies to develop immunohistochemistry-based companion diagnostics for predicting response to therapies targeting RNA processing mechanisms.

• Therapeutic vulnerability assessment: Use DHX34 antibodies to monitor protein levels in relation to drug sensitivity. For instance, DHX34 expression negatively correlates with sensitivity to drugs like BHG712, WZ3105, Methotrexate, COL-3, docetaxel, and linifanib .

• Immunotherapy response prediction: Develop standardized IHC protocols using DHX34 antibodies for predicting immunotherapy outcomes, as lower DHX34 expression predicts more favorable immune checkpoint inhibitor treatment responses in certain cancers .

What are the methodological considerations for investigating DHX34 interactions with RUVBL1-RUVBL2 AAA-ATPases?

To study DHX34's regulation of RUVBL1-RUVBL2 complexes:

• Co-immunoprecipitation approaches: Use DHX34 antibodies for co-IP followed by Western blotting for RUVBL1 and RUVBL2 to confirm interactions. T7-tagged versions of DHX34 have been successfully used in this context .

• In vitro reconstitution assays: Combine purified DHX34 (obtained using immunoaffinity purification) with recombinant RUVBL1-RUVBL2 complexes to study direct effects on ATPase activity.

• Structural analysis considerations: When preparing samples for cryo-EM or other structural studies, consider how antibody binding might affect the conformational changes DHX34 induces in RUVBL1-RUVBL2 complexes.

• Functional validation approaches: Design experiments to test whether DHX34's effect on RUVBL1-RUVBL2 ATPase activity correlates with its function in NMD complex assembly, using antibodies to track complex formation.

• Domain-specific interactions: Use antibodies against specific domains of DHX34 to determine which regions are involved in RUVBL1-RUVBL2 binding and regulation .

How can advanced imaging techniques enhance the study of DHX34 using specific antibodies?

Cutting-edge imaging applications include:

• Super-resolution microscopy: Use fluorescently-labeled DHX34 antibodies for techniques like STORM or PALM to visualize DHX34 distribution at nanoscale resolution in relation to nuclear speckles and other RNA processing bodies.

• Live-cell imaging approaches: Consider developing cell-permeable antibody fragments or nanobodies against DHX34 for live-cell imaging of dynamic RNA processing events.

• Proximity ligation assays (PLA): Combine DHX34 antibodies with antibodies against UPF1, SMG1, or splicing factors to visualize and quantify specific protein-protein interactions in situ at single-molecule resolution.

• Correlative light-electron microscopy (CLEM): Use gold-conjugated DHX34 antibodies to correlate fluorescence localization with ultrastructural context in the nucleus.

• Fluorescence recovery after photobleaching (FRAP): Use fluorescently labeled DHX34 antibodies or anti-tag antibodies with tagged DHX34 to study the dynamics of DHX34 recruitment to active transcription and splicing sites.

What methodological approaches can explore DHX34's role in hematopoietic differentiation and leukemia development?

To investigate DHX34 in hematopoietic contexts:

• Flow cytometry applications: Use DHX34 antibodies in conjunction with hematopoietic lineage markers (CD14, CD71, CD235a) in flow cytometry to correlate DHX34 expression with differentiation status in normal and malignant hematopoiesis .

• Colony formation assays: Monitor DHX34 protein levels using specific antibodies during colony formation assays following DHX34 knockdown or overexpression in CD34+ hematopoietic stem/progenitor cells.

• Patient-derived xenograft models: Use DHX34 antibodies to track protein expression in PDX models of AML/MDS with various genetic backgrounds.

• Differentiation assays: Implement methods to monitor DHX34 during in vitro differentiation protocols for erythroid and myeloid lineages, correlating protein levels with differentiation milestones and the expression of lineage-specific genes.

• Mutation-specific approaches: For studying DHX34 variants found in familial MDS/AML, consider epitope-tagging approaches to distinguish wild-type from mutant proteins when specific antibodies are not available .

How can functional studies of DHX34 mutations found in AML/MDS be designed using available antibodies?

For studying disease-associated DHX34 variants:

• Complementation assays: In DHX34-depleted cells, express wild-type or mutant DHX34 variants and use antibodies to confirm equal expression levels before assessing functional rescue in NMD and splicing assays.

• Protein stability analysis: Use DHX34 antibodies in cycloheximide chase experiments to determine if disease-associated mutations affect protein stability.

• Protein-protein interaction profiling: Compare the interactome of wild-type versus mutant DHX34 using immunoprecipitation with DHX34 antibodies followed by mass spectrometry.

• Subcellular localization studies: Use immunofluorescence with DHX34 antibodies to determine if mutations alter the protein's nuclear localization or subnuclear distribution.

• Patient sample analysis: Develop immunohistochemistry protocols optimized for bone marrow biopsies to detect DHX34 expression and localization in AML/MDS patient samples compared to normal controls.