DDX3Y Antibody

What is DDX3Y and why is it important in research?

DDX3Y (DEAD-box helicase 3 Y-linked) is a member of the DEAD box protein family, characterized by the conserved motif Asp-Glu-Ala-Asp. It functions as an ATP-dependent RNA helicase, playing crucial roles in RNA metabolism and is essential for normal spermatogenesis . DDX3Y is encoded by a gene located on the Y chromosome in the azoospermia factor A region, making it male-specific. Its importance extends to multiple research areas including reproductive biology, immunology, and cancer research, with mutations or deletions in the DDX3Y gene associated with male infertility .

How does DDX3Y differ from its paralog DDX3X?

DDX3Y shares approximately 92% sequence homology with its X-chromosome paralog DDX3X . Despite this high homology, they exhibit distinct expression patterns and functions. While DDX3X is widely expressed and involved in broader cellular functions such as RNA splicing and cell cycle regulation, DDX3Y is predominantly expressed in the testis and specifically in spermatogonia . Recent research has revealed interesting regulatory relationships between these paralogs, with DDX3X capable of suppressing DDX3Y expression through various mechanisms including mRNA destabilization and protein turnover acceleration .

What applications are DDX3Y antibodies suitable for?

DDX3Y antibodies are validated for multiple experimental applications:

Researchers should optimize dilutions for their specific experimental conditions and cell/tissue types .

How should researchers address cross-reactivity concerns when selecting DDX3Y antibodies?

Due to the high sequence homology (~92%) between DDX3Y and DDX3X, cross-reactivity is a significant concern . A thorough analysis of 30 commercial antibodies targeting DDX3Y found that 16 (53%) provided no validation data, and only two included disclaimers warning about potential cross-reactivity with X-chromosome encoded homologs . To address this:

• Select antibodies with validation against both DDX3Y-positive and DDX3Y-negative controls

• Include appropriate controls in experiments (Y-chromosome negative tissues)

• Consider multiple detection methods to confirm specificity

• Validate specificity using knockdown/knockout approaches

• If possible, use antibodies raised against regions with lower homology between DDX3Y and DDX3X

What tissue and cell culture models are optimal for studying DDX3Y expression?

For accurate DDX3Y research, appropriate model selection is critical:

Positive control tissues/cells:

• Human testis tissue (highest expression)

• Male-derived cell lines: DU 145, LNCaP, PC-3 cells (validated by WB)

• Jurkat, Raji, NCI-H929, H1299, HepG2 cells

Negative control tissues/cells:

• Female-derived tissues (lacking Y chromosome)

• Female-derived cell lines (for antibody validation)

Important methodological note: Different buffer systems can affect antibody performance in IHC applications. For DDX3Y detection in testis tissue, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may also be used as an alternative .

How should researchers design experiments to study DDX3Y-DDX3X regulatory relationships?

Based on recent findings about DDX3X regulation of DDX3Y , a comprehensive experimental design should include:

• For transcriptional regulation studies:

  • siRNA-mediated knockdown of DDX3X

  • RT-qPCR measurement of DDX3Y mRNA levels

  • Actinomycin D treatment to assess mRNA stability (half-life calculations)

• For protein-level regulation studies:

  • Overexpression systems with tagged DDX3Y constructs

  • Cycloheximide (CHX) chase assays to measure protein turnover

  • Western blotting with specific antibodies

In a study with HCT116 cells, DDX3X knockdown led to a 44% increase in endogenous DDX3Y protein levels and a 98% increase in DDX3Y mRNA, with the mRNA half-life extended from 3.0 to 4.3 hours .

How can DDX3Y antibodies be utilized in stem cell transplantation and immunotherapy research?

DDX3Y encodes a class I MHC-restricted H-Y antigen recognized by CD8+ T-cells, making it relevant for graft-versus-leukemia (GVL) responses in female-to-male allogeneic hematopoietic cell transplantation . Researchers investigating this area should consider:

• Experimental approach:

  • Detection of DDX3Y-specific CTL responses in transplant recipients

  • Analysis of DDX3Y expression in leukemic stem cells using validated antibodies

  • Correlation of DDX3Y-specific immune responses with clinical outcomes

• Key findings to build upon:

  • DDX3Y-specific CTLs can prevent engraftment of human acute leukemia in NOD/SCID mice

  • Antibody response to DDX3Y minor histocompatibility antigen is induced after allogeneic stem cell transplantation and in healthy female donors

This research direction holds potential for developing targeted immunotherapeutic approaches.

What methodological approaches can resolve contradictory data about DDX3Y expression patterns?

While DDX3Y is primarily described as testis-specific, some studies report detection in other tissues. To resolve such contradictions:

• Comprehensive validation strategy:

  • Employ multiple antibodies targeting different epitopes

  • Use both RNA (RT-PCR) and protein detection methods

  • Include male and female samples as controls

  • Utilize transcript-specific primers to distinguish variant expression

• Transcript variant analysis:

  • Design primer sets spanning from 5'UTR to 3'UTR polyadenylation sites

  • Use nested PCR approaches with "inner primers" to identify specific transcript variants

  • Compare expression patterns across multiple tissues and species

This methodical approach can help distinguish true expression from technical artifacts or cross-reactivity.

How can researchers effectively study the functional consequences of DDX3Y dysregulation?

To investigate DDX3Y's functional roles and the consequences of its dysregulation:

• Knockdown/knockout approaches:

  • siRNA or CRISPR-based targeting of DDX3Y

  • Parallel targeting of DDX3X to study compensatory mechanisms

  • Phenotypic assessment focusing on RNA metabolism and spermatogenesis

• Gene expression analysis:

  • RNA-seq to identify transcripts regulated by DDX3Y

  • RIP (RNA Immunoprecipitation) to identify direct RNA targets

  • Analysis of alternative splicing patterns

• Functional rescue experiments:

  • Complementation with wild-type vs. mutant DDX3Y

  • Cross-complementation with DDX3X to assess functional redundancy

These approaches can provide mechanistic insights into DDX3Y's roles in cellular processes and disease states.

How should researchers troubleshoot unexpected signals when using DDX3Y antibodies?

When encountering unexpected results with DDX3Y antibodies:

• For unexpected bands in female samples:

  • Consider cross-reactivity with DDX3X (92% homology)

  • Verify antibody specificity with peptide competition assays

  • Test multiple antibodies targeting different epitopes

  • Include male positive controls and female negative controls

• For weak or absent signals in male samples:

  • Optimize antigen retrieval (TE buffer pH 9.0 recommended for IHC)

  • Test different antibody dilutions (WB: 1:500-1:1000; IHC: 1:50-1:500)

  • Verify sample integrity (protein degradation)

  • Consider cell/tissue-specific expression patterns (highest in testis)

• For inconsistent results between methods:

  • Compare protein vs. mRNA detection

  • Consider post-translational regulation

  • Evaluate antibody performance across applications

What factors should be considered when interpreting DDX3Y expression data in disease contexts?

When studying DDX3Y in disease contexts:

• Cancer studies:

  • Account for tumor heterogeneity and karyotype alterations

  • Consider Y chromosome loss in male cancers

  • Evaluate DDX3Y mutations and their functional consequences

  • Assess relationship with DDX3X expression (regulatory interplay)

• Infertility research:

  • Correlate DDX3Y expression with spermatogenesis stages

  • Consider transcript variants with tissue-specific functions

  • Evaluate impact of Y chromosome microdeletions

  • Compare with other Y chromosome genes (AZF region)

• Transplantation studies:

  • Account for recipient/donor sex in data interpretation

  • Consider H-Y antigen immunogenicity

  • Evaluate contribution to graft-versus-host disease vs. graft-versus-leukemia effects

This contextual approach helps ensure accurate interpretation of experimental findings.

How can single-cell approaches enhance DDX3Y research?

Single-cell technologies offer new possibilities for studying DDX3Y:

• Single-cell RNA-seq applications:

  • Mapping DDX3Y expression in heterogeneous tissues

  • Identifying rare DDX3Y-expressing cell populations

  • Correlating DDX3Y with cell state and differentiation trajectories

  • Comparing DDX3X/DDX3Y expression at single-cell resolution

• Methodological considerations:

  • Optimize RNA extraction to retain Y-linked transcripts

  • Include spike-in controls to validate detection sensitivity

  • Verify with protein-level detection methods (immunofluorescence)

  • Consider computational approaches to distinguish DDX3Y from DDX3X reads

These approaches can reveal previously unrecognized patterns of DDX3Y expression and regulation.

What novel approaches can improve specificity in DDX3Y antibody-based experiments?

To address the cross-reactivity challenges inherent in DDX3Y research:

• Advanced antibody validation strategies:

  • Use DDX3Y knockout controls generated by CRISPR

  • Employ orthogonal detection methods (MS-based proteomics)

  • Perform systematic epitope mapping

  • Validate across multiple experimental systems

• Technical innovations:

  • Employ proximity ligation assays for increased specificity

  • Consider aptamer-based detection alternatives

  • Develop DDX3Y-specific nanobodies

  • Implement multiplexed detection with DDX3X to assess specificity

Implementing these advanced approaches can significantly improve confidence in experimental findings related to DDX3Y.

DDX3Y Antibody (14041-1-AP) - Proteintech

• Patmore DM, Jassim A, Nathan E, et al. DDX3X Suppresses the Susceptibility of Hindbrain Lineages to Medulloblastoma. Dev Cell. 2020;54(4):455-470.e5. doi:10.1016/j.devcel.2020.05.027

• Mei J, Yan T, Huang Y, et al. A DAZL/CPSF1 axis regulates DDX3Y mRNA translation in human germ cells. Cell Rep. 2019;26(11):2934-2945.e3. doi:10.1016/j.celrep.2019.02.055

• Rosner A, Rinkevich B. The DDX3 subfamily of the DEAD box helicases: divergent roles as unveiled by studying different organisms and in vitro assays. Curr Med Chem. 2007;14(23):2517-2525. doi:10.2174/092986707782023677

• Hoye ML, Stamford A, Ahmed MY, et al. DDX3X loss is a key driver of medulloblastoma with sex-dependent transcriptional effects. Sci Adv. 2021;7(45):eabd3031. doi:10.1126/sciadv.abd3031

• Gong F, Sun G, Zhang Y, et al. Diverse mechanisms of DDX3Y suppression by DDX3X. bioRxiv. 2025;doi:10.1101/2025.02.08.637260

• Wang Q, Peng M, James MA, et al. Survey of commercial antibodies targeting Y chromosome-encoded proteins. bioRxiv. 2023;doi:10.1101/2023.07.26.550552

• Sekiguchi T, Iida H, Fukumura J, et al. Human DDX3Y, the Y-encoded isoform of RNA helicase DDX3, rescues a hamster temperature-sensitive ET24 mutant cell line with a DDX3X mutation. Exp Cell Res. 2004;300(1):213-222. doi:10.1016/j.yexcr.2004.07.005