DPY19L2 Antibody

What is DPY19L2 and what is its biological significance?

DPY19L2 is a multipass transmembrane protein that is highly expressed in the testis. While its function is not fully elucidated in humans, research indicates it plays a crucial role in spermiogenesis (sperm development), particularly in sperm head elongation and acrosome formation . Its ortholog in C. elegans (DPY-19) is involved in establishing cell polarity . Research has demonstrated that homozygous deletion of the DPY19L2 gene causes male infertility, specifically manifesting as globozoospermia (round-headed sperm syndrome) . The protein contains approximately 6-11 transmembrane domains and is present in testicular tissue but absent from mature sperm, suggesting its specific role during spermatogenesis .

What types of DPY19L2 antibodies are available for research applications?

Several formulations of DPY19L2 antibodies are available for research:

• Unconjugated polyclonal antibodies (most common)

• Conjugated versions including:

  • HRP-conjugated for enhanced detection in ELISA and Western blotting

  • FITC-conjugated for fluorescence applications

  • Biotin-conjugated for specialized detection systems

These antibodies target different regions of the DPY19L2 protein, including N-terminal, middle region, and C-terminal epitopes, allowing researchers to select appropriate antibodies based on the experimental design requirements .

What are the optimal sample preparation protocols for DPY19L2 detection?

For optimal DPY19L2 detection:

• Tissue samples:

  • Fresh testicular tissue should be fixed immediately in 4% paraformaldehyde for immunohistochemistry

  • For protein extraction, snap freeze tissue in liquid nitrogen before homogenization

  • Avoid repeated freeze-thaw cycles as noted in handling advice for antibodies

• Western blotting:

  • Use RIPA buffer supplemented with protease inhibitors for protein extraction

  • Load 20-50μg of total protein per lane

  • Use recommended concentration of 1μg/mL for primary antibody incubation

  • Include positive controls (testicular tissue) and negative controls (tissues where DPY19L2 is not expressed)

• Storage of antibodies:

  • Store at 2-8°C for short periods

  • For longer periods, store at -20°C as recommended by suppliers

  • Reconstitute lyophilized antibody powder in distilled water to achieve 1mg/mL concentration in PBS

How can DPY19L2 antibodies be employed to study male infertility mechanisms?

DPY19L2 antibodies offer several methodological approaches to study male infertility:

• Differential expression analysis:

  • Compare DPY19L2 protein expression between fertile and infertile patients using Western blotting

  • Quantify expression levels across different stages of spermatogenesis using immunohistochemistry

• Localization studies:

  • Use immunofluorescence to determine subcellular localization during sperm development

  • Investigate potential interaction with acrosomal proteins and cytoskeletal elements

• Molecular mechanisms:

  • Employ co-immunoprecipitation with DPY19L2 antibodies to identify interaction partners

  • Study DPY19L2's role in cellular polarity based on its evolutionary relationship with C. elegans DPY-19

• Genetic correlation:

  • Correlate DPY19L2 expression with genetic analyses to identify patients with potential mutations or deletions in the DPY19L2 gene

  • Implement antibody-based screening methods for clinical diagnostics of specific forms of male infertility

Research has confirmed that DPY19L2 antibodies detect the protein in human and mouse testis but not in ejaculated human sperm or epididymal mouse sperm, supporting its specific role during spermiogenesis rather than in mature sperm function .

What are the technical challenges in using DPY19L2 antibodies?

Several methodological challenges must be addressed when working with DPY19L2 antibodies:

• Membrane protein challenges:

  • As a multipass transmembrane protein with 6-11 domains, DPY19L2 requires specialized extraction protocols

  • Complete protein denaturation may destroy epitopes while insufficient denaturation may prevent antibody access

• Specificity considerations:

  • The DPY19L gene family includes several paralogs, raising cross-reactivity concerns

  • Validation using blocking peptides is recommended, as noted in product documentation

  • Careful negative controls should include samples from patients with confirmed homozygous DPY19L2 deletions

• Expression timing:

  • Since DPY19L2 expression is stage-specific during spermatogenesis, precise timing of sample collection is critical

  • The protein is absent in mature sperm, requiring testicular biopsy samples for direct detection

• Protocol optimization:

  • Western blotting may require extended transfer times due to DPY19L2's high molecular weight (87 kDa)

  • Membrane blocking and washing steps may need optimization to reduce background

How does DPY19L2 deletion impact spermiogenesis at the molecular level?

DPY19L2 deletion disrupts spermiogenesis through several molecular mechanisms that can be studied using antibody-based techniques:

• Acrosome formation:

  • Immunohistochemistry with DPY19L2 antibodies alongside acrosomal markers reveals the temporal relationship between DPY19L2 expression and acrosome biogenesis

  • Comparative analysis between wild-type and globozoospermic samples demonstrates absence of normal acrosome development in DPY19L2-deficient cells

• Cell polarity disruption:

  • Based on C. elegans ortholog function, DPY19L2 likely influences cell polarity during sperm head formation

  • Co-localization studies with polarity markers can elucidate the mechanism of action

• Cytoskeletal reorganization:

  • DPY19L2 may interact with cytoskeletal elements during sperm head elongation

  • Immunoprecipitation with DPY19L2 antibodies followed by mass spectrometry can identify potential interaction partners

• Genetic correlation:

  • The deletion encompasses approximately 200kb of genomic DNA containing only DPY19L2

  • This deletion results from nonallelic homologous recombination between two highly homologous 28kb low copy repeat sequences flanking the gene

What validation methods should be employed for DPY19L2 antibodies?

Rigorous validation of DPY19L2 antibodies should include:

• Specificity validation:

  • Use blocking peptides specifically designed for the antibody

  • Include positive controls (testicular tissue) and negative controls:

    • Tissues known not to express DPY19L2

    • Samples from patients with confirmed homozygous DPY19L2 deletions

• Cross-reactivity assessment:

  • Test against recombinant proteins of DPY19L family members

  • Evaluate antibody performance in species with divergent DPY19L2 sequences

• Application-specific validation:

  • For Western blotting: confirm band size matches predicted molecular weight (87 kDa)

  • For immunohistochemistry: verify localization patterns match known expression sites

  • For immunoprecipitation: confirm pull-down of specifically interacting proteins

• Reproducibility testing:

  • Test multiple antibody lots to ensure consistent performance

  • Compare polyclonal antibodies from different hosts or sources

How should researchers design experiments to study DPY19L2 in male infertility cases?

Effective experimental design for DPY19L2 research requires:

• Patient selection and categorization:

  • Screen for DPY19L2 deletions in globozoospermia patients

  • Categorize based on genetic analysis: homozygous deletion, heterozygous deletion, point mutations, wild-type

  • Consider family history and parental consanguinity as homozygous deletions are more common in consanguineous families

• Sample collection strategy:

  • Testicular biopsy for direct protein detection (as DPY19L2 is absent in mature sperm)

  • Carefully staged samples to capture DPY19L2 expression during spermatogenesis

  • Paired samples from the same patient when possible (affected/unaffected regions)

• Comprehensive analysis approach:

  • Combine genetic screening, protein expression analysis, and phenotypic characterization

  • Include sperm morphology assessment alongside molecular analyses

  • Consider functional tests like flow cytometry to assess ploidy in affected sperm

• Control selection:

  • Age-matched fertile controls

  • Infertile controls with different etiologies

  • Population-matched controls due to potential ethnic variations in DPY19L2 deletion frequency

What are the key methodological considerations for immunohistochemistry with DPY19L2 antibodies?

For optimal immunohistochemical detection of DPY19L2:

• Tissue preparation:

  • Fresh testicular tissue should be fixed in 4% paraformaldehyde

  • Paraffin embedding followed by 5μm sections is recommended

  • Antigen retrieval is critical due to the multipass transmembrane nature of DPY19L2

• Antibody selection and optimization:

  • Select antibodies targeting extracellular or intracellular domains based on experimental goals

  • Determine optimal antibody concentration (starting with 1μg/mL as recommended)

  • Test multiple antibody clones if available, as epitope accessibility may vary

• Detection system:

  • For fluorescence: consider using FITC-conjugated antibodies for direct detection

  • For chromogenic detection: optimize HRP-conjugated secondary antibodies and substrates

  • Include appropriate controls in each experiment

• Co-localization studies:

  • Pair DPY19L2 detection with markers for acrosome formation

  • Use developmental stage-specific markers to correlate DPY19L2 expression with spermatogenesis phases

How should researchers interpret DPY19L2 antibody results in fertility studies?

Proper interpretation of DPY19L2 antibody results requires:

• Expression pattern analysis:

  • DPY19L2 should be detected in testicular tissue but not in mature sperm

  • Abnormal or absent expression patterns may indicate pathology

  • Correlation between protein expression and genetic status is essential for comprehensive understanding

• Comparative analysis:

  • Compare results between fertile controls and patients with known fertility issues

  • Consider results in context of sperm morphology and function tests

  • Integrate findings with genetic analyses (presence of DPY19L2 deletions or mutations)

• Statistical considerations:

  • Quantify protein expression levels when possible

  • Apply appropriate statistical tests based on sample distribution

  • Consider potential confounding factors like age, other medical conditions, and technical variables

• Clinical correlation:

  • Connect molecular findings with clinical presentation

  • Consider family history, particularly consanguinity which increases risk of homozygous deletions

  • Understand that DPY19L2 deletions are specifically associated with globozoospermia

What are common technical issues with DPY19L2 antibodies and their solutions?

Researchers frequently encounter these technical challenges:

• High background in Western blots:

  • Increase blocking time or concentration (5% BSA often preferable to milk for membrane proteins)

  • Optimize antibody concentration (starting with recommended 1μg/mL)

  • Increase wash duration and volume

  • Use blocking peptides to confirm specificity

• No signal detection:

  • Confirm sample type contains DPY19L2 (testis vs. mature sperm)

  • Verify protein extraction protocol is suitable for membrane proteins

  • Try multiple antibodies targeting different epitopes

  • Check for genetic deletions that would eliminate the target protein

• Multiple bands or unexpected band sizes:

  • Verify predicted molecular weight (87 kDa for full-length protein)

  • Consider post-translational modifications or proteolytic processing

  • Implement additional protease inhibitors during sample preparation

  • Use blocking peptides to identify specific vs. non-specific bands

• Inconsistent results:

  • Standardize sample collection and processing protocols

  • Maintain consistent antibody lots when possible

  • Include positive and negative controls in each experiment

  • Document all protocol variations for troubleshooting

How can DPY19L2 antibodies contribute to the development of diagnostic tools for male infertility?

DPY19L2 antibodies offer potential for diagnostic applications:

• Immunohistochemical screening:

  • Development of standardized IHC protocols for testicular biopsy evaluation

  • Creation of diagnostic algorithms combining DPY19L2 expression with morphological assessment

• Biomarker development:

  • Exploration of DPY19L2 fragments or interacting proteins as non-invasive biomarkers

  • Correlation of protein expression patterns with specific infertility phenotypes

• Complementary diagnostic approaches:

  • Integration with genetic screening for DPY19L2 deletions

  • Development of comprehensive diagnostic panels combining protein and genetic analyses

  • Establishment of standardized reference ranges for DPY19L2 expression

• Predictive testing:

  • Investigation of partial defects in DPY19L2 expression as potential predictors of subfertility

  • Correlation of expression levels with severity of globozoospermia and fertility outcomes

What future research directions involve DPY19L2 antibodies?

Emerging research opportunities include:

• Functional studies:

  • Investigation of DPY19L2's role in cellular polarity during spermiogenesis

  • Exploration of potential enzymatic activities based on structural analysis

  • Examination of evolutionary conservation across species

• Therapeutic development:

  • Screening for compounds that can rescue or bypass DPY19L2 deficiencies

  • Development of targeted interventions for specific forms of male infertility

  • Personalized medicine approaches based on DPY19L2 status

• Expanded methodologies:

  • Development of super-resolution microscopy techniques for detailed localization

  • Implementation of proximity ligation assays to identify interaction partners

  • Application of CRISPR/Cas9 models combined with antibody-based detection systems

• Comprehensive mapping:

  • Detailed temporal expression mapping during spermatogenesis

  • Comparative analysis across species to understand evolutionary conservation

  • Integration with other reproductive biology research to build comprehensive models of spermiogenesis