DDX24 Antibody

What is DDX24 and what cellular functions has it been linked to?

DDX24 is an 859 amino acid ATP-dependent RNA helicase belonging to the DEAD-box family of helicases. Research has demonstrated DDX24's involvement in multiple biological processes including:

• Negative regulation of cytosolic RNA-mediated innate immune responses

• Promotion of metastasis in non-small cell lung cancer (NSCLC)

• Vascular smooth muscle cell (VSMC) development and cell cycle regulation

• Liquid-liquid phase separation, particularly relevant to nucleolar function

• Increased expression in Alzheimer's disease brain tissues

Unlike other DEAD-box helicases, DDX24 possesses several potential interferon-regulated transcription sites in its promoter region, including STAT1 and IRF7 binding sites, similar to RIG-I, MDA5, and LGP2 .

What is the discrepancy between calculated and observed molecular weight of DDX24?

While the calculated molecular weight of DDX24 is 96 kDa based on amino acid sequence, the observed molecular weight in SDS-PAGE is consistently around 120 kDa . This discrepancy is likely due to post-translational modifications or the protein's structural properties. When validating DDX24 antibody specificity, researchers should expect to observe a band at approximately 120 kDa, and knockdown experiments should demonstrate reduced intensity at this position. Multiple commercial antibodies consistently report this observation.

What domains are important for DDX24 function and antibody epitope selection?

DDX24 contains:

• An N-terminal region rich in glutamic acid and lysine residues

• A DExD/H box helicase ATP binding domain (important for FADD association)

• Low-confidence regions spanning positions 250-380 associated with intrinsical disorder

• Intrinsically disordered regions (IDRs) in the N-terminal domain (NTD), C-terminal domain (CTD), and the low-confidence region that contribute to its phase separation properties

When selecting antibodies, consider whether targeting specific domains (like the ATP-binding domain) may interfere with protein-protein interactions in co-immunoprecipitation experiments.

What applications are validated for commercial DDX24 antibodies?

Based on validation data from multiple antibody providers, DDX24 antibodies have been successfully used in:

*Ranges vary by specific antibody product

How should I optimize sample preparation for DDX24 detection in western blotting?

For optimal DDX24 detection in western blot:

• Prepare cell lysates in RIPA buffer supplemented with protease inhibitors

• Load 20-40 μg of total protein per lane

• Use a 4-12% gradient gel for better resolution of high molecular weight proteins

• Transfer to PVDF membrane at low voltage (30V) overnight for larger proteins

• Block membranes with 5% non-fat milk or BSA in TBST

• Incubate with primary antibody at recommended dilution (1:1000-1:50000, depending on specific antibody)

• Validate specificity using DDX24 knockdown controls as demonstrated in HUVECs

What are the recommended antigen retrieval methods for DDX24 immunohistochemistry?

For optimal IHC results:

• Primary recommendation: Use TE buffer pH 9.0 for antigen retrieval

• Alternative method: Citrate buffer pH 6.0 can also be effective

• Block background with peroxidase treatment at room temperature for 10 minutes

• Incubate overnight at 4°C with primary anti-DDX24 antibody (1:100-1:800 dilution)

• Include negative control samples where primary antibody is omitted

• Validate antibody specificity using siRNA silencing in appropriate cell types

How can I design experiments to study DDX24's role in innate immune responses?

DDX24 negatively regulates cytosolic RNA-mediated innate immune responses through several mechanisms:

• RNA competition experiments:

  • Determine if DDX24 competes with RIG-I for RNA binding using in vitro binding assays

  • Use biotinylated polyI:C or ssRNA to precipitate DDX24 from cell lysates

  • Perform RNA pull-down with the VSV-G gene transcript to study sequence-specific interactions

• Signaling pathway analysis:

  • Employ luciferase reporter assays with IFNβ promoter following polyI:C transfection

  • Compare wild-type cells to DDX24 knockdown cells to assess enhanced type I IFN production

  • Measure immune-related gene expression changes using RT-qPCR or microarray analysis

• Viral infection models:

  • Use VSV expressing luciferase (VSV-Luc) or GFP (VSV-GFP) in DDX24-depleted cells

  • Monitor viral replication through luciferase activity, viral titers, or fluorescence

  • Include RIG-I knockdown as a positive control for enhanced viral replication

What methods can effectively analyze DDX24's liquid-liquid phase separation properties?

DDX24 has been shown to undergo liquid-liquid phase separation (LLPS), which may be relevant to its biological function:

• In vitro phase separation assays:

  • Purify full-length DDX24WT and DDX24E271K proteins labeled with fluorescent markers (e.g., Alexa Fluor 488)

  • Test LLPS under varying conditions:

    • Low ionic strength buffers

    • Crowding agents

    • Different protein concentrations

  • Observe formation of condensates, fibers, or gel-like structures

• Structural and sequence analysis:

  • Use protein disorder predictors (e.g., IUPRED) to identify intrinsically disordered regions

  • Visualize DDX24 structure using AlphaFold2 to identify low-confidence/disordered regions

  • Analyze how mutations (e.g., E271K) within IDRs affect phase separation properties

• Live-cell imaging:

  • Express fluorescently tagged DDX24 in relevant cell types

  • Monitor condensate formation, fusion events, and dynamic properties

  • Use FRAP (Fluorescence Recovery After Photobleaching) to measure protein mobility within condensates

How can I investigate DDX24's role in cancer progression and metastasis?

To study DDX24's contribution to cancer progression, particularly in NSCLC:

• Gene expression modulation:

  • Establish stable DDX24 knockdown cell lines using shRNA:

    • shRNA-1: 5′-GCAGUCAAGCUGUGGCAAATT-3′

    • shRNA-2: 5′-GGGAGAAACCUGUUCCCAATT-3′

  • Transfect cells with DDX24-CMV-GFP-Puro plasmid for overexpression

  • Confirm knockdown/overexpression efficiency by western blot and RT-qPCR

• Migration and invasion assays:

  • Perform wound healing assays to assess cell migration

  • Use transwell chambers with or without Matrigel coating to evaluate invasion

  • Compare results between DDX24-modulated cells and controls

• In vivo metastasis models:

  • Inject DDX24-modified cancer cells into animal models

  • Monitor metastatic spread using in vivo imaging systems

  • Analyze metastatic tissues by histology and immunohistochemistry

• Interaction partner analysis:

  • Investigate DDX24's interaction with RPL5, which has been implicated in its pro-metastatic function in NSCLC

  • Use co-immunoprecipitation followed by western blotting or mass spectrometry to identify novel interaction partners

What approaches can reveal DDX24's role in vascular development?

DDX24 is essential for vascular smooth muscle cell (VSMC) function and embryonic vascular development:

• Conditional knockout models:

  • Generate tissue-specific DDX24 knockout mice (e.g., using Tagln-Cre mice crossed with Ddx24 flox/flox mice)

  • Analyze vascular development by stereomicroscopy and immunofluorescence staining

  • Examine extraembryonic tissues for abnormal vascular remodeling

• Cell cycle and proliferation analysis:

  • Perform flow cytometry to assess cell cycle distribution in DDX24-depleted VSMCs

  • Use BrdU or EdU incorporation assays to measure proliferation

  • Evaluate DNA damage markers to correlate with cell cycle arrest

• RNA-protein interaction studies:

  • Conduct RNA immunoprecipitation coupled with quantitative real-time PCR

  • Identify mRNAs stabilized by DDX24, such as FANCA (FA complementation group A)

  • Perform RNA stability experiments following actinomycin D treatment

• Rescue experiments:

  • Overexpress FANCA in DDX24-deficient cells to assess functional rescue

  • Analyze whether DNA damage and cell cycle defects are reversed

How should I address inconsistencies in DDX24 antibody staining patterns?

When encountering variable DDX24 staining patterns:

• Subcellular localization verification:

  • DDX24 localizes to both nucleus and cytoplasm under basal conditions

  • Following poly I:C treatment, elevated cytoplasmic DDX24 is observed at 6 and 9 hours

  • Confirm localization patterns using subcellular fractionation and immunoblotting

  • For immunofluorescence, use nuclear markers (DAPI) and cytoplasmic markers for co-localization studies

• Antibody validation strategies:

  • Perform siRNA/shRNA knockdown of DDX24 and confirm reduced staining

  • Use multiple antibodies targeting different epitopes of DDX24

  • Include positive control cell lines known to express DDX24 (HeLa, A549, THP-1)

• Fixation optimization:

  • Test different fixation methods (paraformaldehyde, methanol, acetone)

  • Optimize antigen retrieval conditions (pH, temperature, duration)

  • Adjust blocking conditions to reduce background

What controls should I include when studying DDX24's functional effects?

To ensure robust data interpretation when studying DDX24:

• Essential controls for knockdown experiments:

  • Non-targeting shRNA/siRNA control

  • Multiple shRNA/siRNA sequences targeting different regions of DDX24 to rule out off-target effects

  • Rescue experiments with shRNA-resistant DDX24 expression constructs

  • Quantitative assessment of knockdown efficiency by western blot and RT-qPCR

• Critical controls for overexpression studies:

  • Empty vector controls

  • Tagged DDX24 expression verification by western blot

  • Wild-type vs. mutant DDX24 comparisons (e.g., E271K mutation)

  • Dose-dependent analysis to avoid artifacts from excessive overexpression

• Pathway-specific controls:

  • For innate immunity studies: RIG-I knockdown as a comparative control

  • For cell cycle studies: DNA damage inducers (e.g., etoposide)

  • For RNA stability studies: Actinomycin D treatment time courses

How can I detect and study nascent RNA interactions with DDX24?

To investigate DDX24's association with nascent RNA:

• EU incorporation assay protocol:

  • Seed cells in glass-bottom confocal dishes (10,000 cells per dish)

  • Twenty-four hours later, treat cells with desired chemicals

  • Incubate with 0.5 mM ethyl uridine (EU) for 1 hour

  • Wash thoroughly and fix cells after EU incubation

  • Detect EU using Cell-Light EU Apollo-488 RNA Imaging Kit

  • Co-stain with DDX24 antibody to assess co-localization

• RNA immunoprecipitation:

  • Cross-link RNA-protein complexes with formaldehyde or UV

  • Lyse cells and perform immunoprecipitation with DDX24 antibody

  • Extract and analyze associated RNAs by RT-qPCR or RNA-seq

  • Include IgG control and DDX24 knockdown samples as negative controls

• RNA pull-down:

  • Synthesize biotinylated RNA probes of interest

  • Incubate with cell lysates containing DDX24

  • Precipitate RNA-protein complexes with streptavidin beads

  • Detect DDX24 by western blotting

  • Compare binding affinities between different RNA sequences

How might DDX24 be involved in neurodegenerative disorders?

Recent research has identified increased DDX24 in Alzheimer's disease brains:

• Temporal expression analysis:

  • AppNL-F mouse models show that DDX24 increases before amyloid pathology or memory impairment

  • Use immunohistochemistry at different disease stages to track progression

  • Employ antibody HPA 002554 (1:100 dilution) for reliable detection

• Validation approaches:

  • Confirm antibody specificity through siRNA silencing in primary neuronal cultures

  • Include negative controls by omitting primary antibody

  • Perform peroxidase blocking (10 min at room temperature) followed by PBS-T washing

• Mechanistic studies:

  • Investigate DDX24's potential role in RNA metabolism disruption in neurodegeneration

  • Explore interactions with known Alzheimer's-associated proteins

  • Examine DDX24's impact on stress granule formation in neuronal models

How can I study DDX24's interaction with other proteins?

To characterize DDX24's protein-protein interactions:

• Co-immunoprecipitation and mass spectrometry:

  • Use anti-DDX24 antibody (Bethyl, A300-698A) with Pierce™ Co-Immunoprecipitation Kit

  • Include normal rabbit IgG as a control

  • Resolve pull-down proteins via SDS-PAGE followed by silver staining

  • Identify interacting partners by mass spectrometry

  • Use AP-MS (affinity purification-mass spectrometry) scoring via the REPRINT pipeline

• Yeast two-hybrid screening:

  • Use DDX24 as bait to identify novel interacting partners

  • Confirm specific interactions by reverse Y2H (using identified proteins as bait)

  • Validate interactions in mammalian cells by co-IP

• Domain mapping:

  • Generate DDX24 domain truncation constructs

  • Perform co-IP experiments to determine which domains mediate specific interactions

  • For example, the amino terminal region containing the DExD/H box helicase ATP binding domain mediates FADD association