DTX36 Antibody

What is DHX36 and what are its primary functions?

DHX36 is a multifunctional ATP-dependent helicase that primarily unwinds G-quadruplex (G4) structures in nucleic acids. It plays critical roles in genomic integrity, gene expression regulation, and functions as a sensor to initiate antiviral responses . The protein binds with high affinity to and unwinds G4 structures formed in both DNA and RNA molecules (G4-DNA and G4-RNA), making it essential for numerous cellular processes . These G4 structures consist of helical arrangements containing guanine tetrads that DHX36 specifically targets and resolves.

What biological processes involve DHX36?

DHX36 participates in several crucial biological processes:

• Genomic integrity maintenance: Resolves G4 structures that could otherwise lead to genomic instability

• Transcriptional regulation: Resolves G4-DNA structures in gene promoters (including YY1, KIT/c-kit, and ALPL) to positively regulate their expression

• Post-transcriptional regulation: Unwinds G4-RNA structures in the 3'-UTR polyadenylation sites of pre-mRNAs, such as TP53, stimulating their 3'-end processing in response to DNA damage

• Telomere maintenance: Converts G4-RNA structures in telomerase RNA template components into double-stranded RNA to promote P1 helix formation

• MicroRNA processing: Binds to pre-miRNA terminal loops, regulating their transport and contributing to the control of dendritic spine morphogenesis

What applications is the DHX36 antibody suitable for?

The rabbit polyclonal DHX36 antibody (ab70269) has been validated for multiple research applications, including:

• Immunoprecipitation (IP)

• Western Blotting (WB)

• Immunohistochemistry on paraffin-embedded tissues (IHC-P)

The antibody has been cited in 14 scientific publications, demonstrating its reliability and acceptance in the research community . It has been confirmed to react with human and mouse samples, making it suitable for comparative studies across these species.

How does DHX36 specificity for G-quadruplex structures impact experimental design?

When designing experiments to study DHX36 function, researchers must account for its high specificity for G-quadruplex structures. The unique binding affinity of DHX36 for G4 structures requires careful consideration of nucleic acid sequence contexts in experimental systems.

When analyzing DHX36 interactions with potential target sequences, researchers should:

• Identify putative G-quadruplex forming sequences (G-rich regions with potential to form G-tetrads)

• Confirm G4 structure formation using techniques such as circular dichroism spectroscopy

• Analyze DHX36 binding and unwinding activity using both G4-forming and control (non-G4) sequences

• Consider the impact of different cations (particularly potassium) on G4 stability during buffer preparation

• Account for potential competition between DHX36 and other G4-binding proteins in cellular contexts

This specificity also offers opportunities for targeted experiments exploring the role of G4 structures in specific genomic contexts, such as promoter regions of YY1, KIT, and ALPL genes .

How can researchers distinguish between DHX36 effects on DNA versus RNA G-quadruplexes?

Distinguishing DHX36 activity on DNA versus RNA G-quadruplexes requires specialized experimental approaches:

• Substrate specificity assays: Compare DHX36 binding affinity and unwinding rates between equivalent G4-DNA and G4-RNA sequences using purified recombinant protein and synthetic oligonucleotides.

• Nuclease protection assays: Utilize DNA-specific (DNase I) or RNA-specific (RNase A) nucleases to selectively degrade non-G4 structures while protecting DHX36-bound substrates.

• Cross-linking immunoprecipitation (CLIP) approaches:

  • CLIP-seq for RNA interactions

  • ChIP-seq for DNA interactions
    These can be performed in parallel to map genome-wide DNA versus RNA interactions.

• Cellular compartment analysis: Examine DHX36 distribution between nucleus (primarily DNA interactions) and cytoplasm (primarily RNA interactions) using fractionation followed by Western blotting.

• Mutational analysis: Create DHX36 mutants with selective defects in either DNA or RNA G4 binding to distinguish substrate-specific activities.

What are the implications of DHX36's role in telomerase RNA template component (TERC) for aging and cancer research?

DHX36's critical function in telomerase RNA processing positions it as a potential target in both aging and cancer research:

• Cancer implications: DHX36 converts G4-RNA structures in telomerase RNA template components (TERC) into double-stranded RNA to promote P1 helix formation, ensuring accurate reverse transcription . Cancer cells typically upregulate telomerase to maintain telomere length and achieve replicative immortality. DHX36 inhibition could potentially disrupt this process by preventing proper TERC processing.

• Aging research: Telomere shortening is a hallmark of cellular aging. DHX36's role in TERC processing may impact the efficiency of telomere maintenance in somatic cells. Researchers studying aging should consider DHX36 expression and activity when investigating telomere dynamics in senescent cells.

• Experimental considerations: Researchers examining DHX36 in these contexts should:

  • Monitor telomere length using q-FISH or qPCR-based methods

  • Assess telomerase activity via TRAP assays

  • Examine TERC processing and structure using RNA structure probing techniques

  • Analyze correlations between DHX36 expression levels and telomere maintenance in various cell types

What are the optimal conditions for using DHX36 antibody in Western blotting?

When using DHX36 antibody (ab70269) for Western blotting, researchers should consider the following technical parameters for optimal results:

• Sample preparation:

  • Extract proteins using RIPA buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylation-dependent activities of DHX36

  • Denature samples in Laemmli buffer with DTT at 95°C for 5 minutes

• Gel electrophoresis:

  • Use 8-10% SDS-PAGE gels (DHX36 is approximately 110 kDa)

  • Load 20-30 μg of total protein per lane for cell lysates

  • Include positive control lysates from cells known to express DHX36

• Transfer conditions:

  • Wet transfer to PVDF membrane at 100V for 90 minutes with cooling

  • Alternatively, semi-dry transfer at 25V for 30 minutes for faster results

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Dilute primary antibody 1:1000 in blocking solution

  • Incubate with primary antibody overnight at 4°C with gentle agitation

  • Wash 3 times with TBST, 5 minutes each

  • Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

  • Wash 3 times with TBST, 5 minutes each

• Detection:

  • Use enhanced chemiluminescence (ECL) substrate

  • Expect band at approximately 110 kDa corresponding to DHX36

How should researchers approach DHX36 immunoprecipitation for studying associated RNA or DNA?

For effective immunoprecipitation of DHX36 and its associated nucleic acids:

• Cross-linking optimization:

  • For RNA interactions: Use 0.1-0.3% formaldehyde or UV cross-linking (254 nm)

  • For DNA interactions: Use 1% formaldehyde for 10 minutes at room temperature

• Lysis conditions:

  • Use mild lysis buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA)

  • Include RNase inhibitors when studying RNA interactions

  • Add benzonase for DNA studies to reduce background

• Antibody selection:

  • Use 5 μg of DHX36 antibody (ab70269) per IP reaction

  • Pre-clear lysate with protein A/G beads before adding antibody

  • Include IgG control IP to identify non-specific binding

• Washing and elution:

  • Perform stringent washes (at least 5) with lysis buffer

  • For DNA studies: Include high-salt wash steps (up to 500 mM NaCl)

  • For RNA studies: Keep salt concentration lower (150 mM NaCl maximum)

  • Elute bound complexes with sample buffer for protein analysis or by proteinase K digestion for nucleic acid analysis

• Recovery and analysis of bound nucleic acids:

  • Extract RNA using TRIzol or similar reagent

  • Extract DNA using phenol-chloroform

  • Analyze by qPCR, sequencing, or structure-specific assays for G-quadruplexes

What controls should be included when studying DHX36 in cellular research systems?

When conducting research on DHX36, several critical controls should be included:

• Expression controls:

  • Positive control: Cell lines with confirmed high DHX36 expression (e.g., HeLa cells)

  • Negative control: DHX36 knockdown/knockout cells generated via siRNA or CRISPR-Cas9

  • Rescue control: DHX36-deficient cells with reintroduced wild-type DHX36

• Functional controls:

  • Catalytic-dead mutant: DHX36 with mutations in the ATPase domain to eliminate helicase activity

  • G4-binding mutant: DHX36 with mutations in the G4-binding domain to eliminate G4 recognition

  • Substrate controls: G4-forming sequences versus matched sequences unable to form G4 structures

• Localization controls:

  • Nuclear marker (e.g., HDAC1) for nuclear DHX36 functions

  • Cytoplasmic marker (e.g., GAPDH) for cytoplasmic DHX36 functions

  • Fluorescent tagged DHX36 with subcellular markers for colocalization studies

• Antibody validation controls:

  • Secondary antibody only control to assess non-specific binding

  • Pre-absorption control with immunizing peptide to confirm specificity

  • Multiple antibodies targeting different DHX36 epitopes to confirm observations

What are common issues encountered in DHX36 immunohistochemistry and how can they be addressed?

When performing immunohistochemistry with DHX36 antibody, researchers may encounter several challenges:

• High background staining:

  • Problem: Non-specific binding of primary or secondary antibodies

  • Solution: Increase blocking time (2-3 hours), use higher concentration of blocking protein (5-10% normal serum), include 0.1% Triton X-100 in blocking solution to reduce non-specific binding

• Weak or absent signal:

  • Problem: Insufficient antigen retrieval or antibody concentration

  • Solution: Optimize antigen retrieval methods (try citrate buffer pH 6.0 or EDTA buffer pH 9.0), increase antibody concentration, extend primary antibody incubation to overnight at 4°C

• Inconsistent staining between samples:

  • Problem: Variability in fixation or processing

  • Solution: Standardize fixation protocols (10% neutral buffered formalin for 24 hours), ensure consistent section thickness (4-5 μm), process all samples simultaneously

• Nuclear versus cytoplasmic localization discrepancies:

  • Problem: DHX36 can localize to both compartments depending on cellular context

  • Solution: Use confocal microscopy with Z-stack imaging to precisely determine localization, include co-staining with nuclear and cytoplasmic markers

• Tissue-specific optimization:

  • Problem: Different tissues may require different protocols

  • Solution: Perform titration experiments for each new tissue type, adjust antigen retrieval time based on tissue density

How can researchers overcome variability in DHX36 detection across different cell lines?

Variability in DHX36 detection across cell lines represents a common challenge that can be addressed through systematic optimization:

• Expression level differences:

  • Problem: DHX36 expression varies significantly between cell types

  • Solution: Adjust loading amounts based on preliminary expression screening, use housekeeping genes with similar expression levels across cell types for normalization

• Post-translational modification variations:

  • Problem: DHX36 may undergo different modifications affecting antibody recognition

  • Solution: Use phosphatase inhibitors consistently, consider using multiple antibodies recognizing different epitopes, perform immunoprecipitation followed by mass spectrometry to identify modifications

• Extraction efficiency differences:

  • Problem: DHX36 may be differentially extracted from different cell types

  • Solution: Compare multiple lysis protocols (RIPA, NP-40, sonication-based), include benzonase to release chromatin-bound protein, optimize lysis time and conditions for each cell type

• Interference from binding partners:

  • Problem: Cell-specific DHX36 complexes may mask antibody binding sites

  • Solution: Include brief denaturation step, test different detergent concentrations, use epitope retrieval techniques similar to those in immunohistochemistry

• Cross-reactivity concerns:

  • Problem: Antibody may recognize related helicases in certain cell contexts

  • Solution: Confirm specificity using DHX36 knockout cells for each cell line, perform peptide competition assays, validate with orthogonal methods (e.g., mass spectrometry)

What are emerging research areas involving DHX36 that researchers should consider?

Based on current understanding of DHX36 function, several promising research directions warrant investigation:

• Role in antiviral immunity:

  • DHX36 functions as a sensor to initiate antiviral responses

  • Researchers should investigate its interactions with viral RNA containing G4 structures

  • Potential for therapeutic targeting in viral infections

• DHX36 in neurodegenerative disorders:

  • G4 structures are prevalent in genes associated with neurodegeneration

  • DHX36's role in resolving these structures may impact disease progression

  • Investigation of DHX36 expression and function in neurodegenerative disease models

• Cancer therapeutic applications:

  • DHX36's influence on oncogene expression through G4 resolution

  • Potential for synthetic lethality approaches in tumors with altered G4 metabolism

  • Development of small molecule DHX36 inhibitors as possible therapeutics

• Roles in RNA stress granule dynamics:

  • G4 structures in mRNAs can influence stress granule formation

  • DHX36 may regulate stress response through unwinding of G4-RNAs

  • Investigation of DHX36 localization and function during cellular stress

• Interplay with other G4 binding proteins:

  • Cooperative or competitive interactions with other G4 binding factors

  • Regulatory networks controlling G4 structure resolution

  • Temporal dynamics of G4 formation and resolution in different cellular contexts

How might single-cell approaches advance understanding of DHX36 function?

Single-cell technologies offer powerful approaches to understand DHX36 function with unprecedented resolution:

• Single-cell RNA sequencing applications:

  • Examine cell-to-cell variability in DHX36 expression

  • Correlate DHX36 levels with expression of G4-regulated genes

  • Identify cell-type specific roles in heterogeneous tissues

• Single-cell ATAC-seq combined with DHX36 analysis:

  • Map chromatin accessibility at G4-containing promoters

  • Correlate with DHX36 expression levels

  • Identify cell states where DHX36 activity may be particularly important

• In situ approaches:

  • Develop G4-structure specific probes for visualization in intact cells

  • Correlate G4 abundance with DHX36 localization at single-cell level

  • Track dynamic changes in G4 structures during cell cycle or differentiation

• Single-molecule imaging:

  • Track individual DHX36 molecules in living cells

  • Measure dwelling time at specific genomic loci

  • Determine kinetics of G4 resolution in different cellular contexts

• Single-cell proteomics:

  • Quantify DHX36 protein levels alongside interacting partners

  • Identify cell-specific DHX36 complexes

  • Correlate post-translational modifications with functional outcomes