SPATA19 Antibody

What is SPATA19 and what is its biological function?

SPATA19 (spermatogenesis associated 19), also known as spergen1 (spermatogenic cell-specific gene 1 protein), CT132, or SPAS1, is a 167 amino acid mitochondrial outer membrane protein primarily involved in spermiogenesis. It is specifically expressed in testis tissue and is encoded by a gene that maps to human chromosome 11, which comprises approximately 4% of human genomic DNA and is considered a gene and disease association dense chromosome . The protein plays a crucial role in sperm development, particularly in the differentiation process of spermatids into mature spermatozoa. Research indicates that SPATA19's localization to the mitochondrial outer membrane suggests its involvement in mitochondrial function during sperm maturation and possibly in regulating energy metabolism essential for sperm motility .

What is the molecular structure and characteristics of SPATA19?

SPATA19 is characterized as a 167 amino acid protein with a calculated molecular weight of 19 kDa . The observed molecular weight in experimental settings consistently confirms this calculation at approximately 19 kDa . The protein is primarily associated with the mitochondrial outer membrane, which aligns with its proposed function in sperm development . The gene encoding SPATA19 is found on chromosome 11, with GenBank accession number BC058039 . The protein's UniProt ID is Q7Z5L4 . SPATA19's structure includes specific domains that facilitate its interaction with mitochondrial components, though the detailed tertiary structure requires further characterization through crystallography studies.

What types of SPATA19 antibodies are available for research purposes?

Current research primarily utilizes polyclonal antibodies against SPATA19, with rabbit being the predominant host species. These antibodies are generated using fusion proteins of human SPATA19 as immunogens . The most commonly available antibodies include:

These antibodies undergo antigen affinity purification to ensure specificity and are generally provided in buffer solutions containing preservatives like sodium azide and stabilizers such as glycerol .

How should researchers determine the optimal SPATA19 antibody for their experimental design?

When selecting a SPATA19 antibody, researchers should consider multiple factors based on their experimental requirements. First, determine the species compatibility needed for your study - available antibodies show reactivity with human, mouse, and rat samples . Second, consider the intended application, as different antibodies perform with varying efficiencies in Western blot (WB), immunohistochemistry (IHC), or immunofluorescence (IF) techniques .

The application-specific dilution recommendations are critical for optimal results:

Always perform preliminary titration experiments with your specific samples to determine optimal antibody concentration. For challenging applications or when studying tissues with low SPATA19 expression, selecting antibodies with published validation in similar experimental contexts can significantly improve success rates .

What are the optimal protocols for SPATA19 detection by Western blotting?

For effective Western blot detection of SPATA19, researchers should implement a methodical approach. Begin with protein extraction from tissues known to express SPATA19, particularly testis tissue from human, mouse, or rat sources . A standard protocol involves:

• Sample preparation: Homogenize tissue in RIPA buffer supplemented with protease inhibitors.

• Protein quantification: Use Bradford or BCA assay to normalize loading.

• SDS-PAGE: Load 20-50 μg of protein per lane on 12-15% gels (optimal for detecting the 19 kDa SPATA19 protein).

• Transfer: Use PVDF membranes for better protein retention.

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

• Primary antibody incubation: Dilute SPATA19 antibody to 1:500-1:2000 in blocking buffer and incubate overnight at 4°C .

• Washing: 3-5 times with TBST, 5 minutes each.

• Secondary antibody: Anti-rabbit HRP-conjugated antibody (1:5000) for 1 hour at room temperature.

• Development: Use enhanced chemiluminescence detection.

For challenging samples, increasing the primary antibody concentration or extending incubation time may improve signal detection. Because SPATA19 is primarily expressed in testis, using testis tissue as a positive control is essential for validating experimental results .

What are the recommended protocols for immunohistochemical detection of SPATA19?

For immunohistochemical detection of SPATA19, researchers should follow these methodological guidelines to achieve optimal staining:

• Tissue preparation: Fix tissues in 10% neutral buffered formalin and embed in paraffin.

• Sectioning: Prepare 4-6 μm sections on positively charged slides.

• Deparaffinization: Clear sections with xylene and rehydrate through graded alcohols.

• Antigen retrieval: This is critical for SPATA19 detection. Use TE buffer pH 9.0 as recommended, or alternatively, citrate buffer pH 6.0 .

• Endogenous peroxidase blocking: Treat with 3% hydrogen peroxide for 10 minutes.

• Protein blocking: Use 5% normal serum for 30 minutes.

• Primary antibody: Apply SPATA19 antibody at 1:20-1:200 dilution and incubate overnight at 4°C .

• Detection system: Use polymer-based detection systems for enhanced sensitivity.

• Chromogen development: DAB substrate for 3-5 minutes or until optimal signal appears.

• Counterstaining: Hematoxylin for nuclear visualization.

Human testis tissue serves as the primary positive control, but SPATA19 can also be detected in human liver, lung, ovary, placenta, skin, and spleen tissues with appropriate optimization . For challenging tissues, extending the primary antibody incubation time or increasing concentration within the recommended range may enhance detection sensitivity.

How can immunofluorescence be optimized for SPATA19 detection in reproductive tissues?

Immunofluorescence detection of SPATA19 requires careful optimization, particularly when examining its subcellular localization in the mitochondrial outer membrane. The following protocol maximizes detection sensitivity:

• Sample preparation: For fresh tissue, fix in 4% paraformaldehyde for 24 hours; for frozen sections, cut at 5-8 μm thickness.

• Permeabilization: Treat with 0.2% Triton X-100 for 10 minutes to access intracellular antigens.

• Blocking: Use 5% BSA in PBS for 1 hour at room temperature.

• Primary antibody: Apply SPATA19 antibody diluted 1:50-1:500 in blocking solution and incubate overnight at 4°C .

• Washing: Perform 3-5 washes with PBS containing 0.1% Tween-20.

• Secondary antibody: Use fluorophore-conjugated (Alexa Fluor 488 or 594) anti-rabbit antibody diluted 1:500 for 1 hour at room temperature.

• Nuclear counterstaining: DAPI (1 μg/ml) for 5 minutes.

• Mounting: Use anti-fade mounting medium to prevent photobleaching.

For co-localization studies confirming mitochondrial association, combine SPATA19 antibody with mitochondrial markers such as MitoTracker or antibodies against COX IV. Mouse testis tissue serves as an excellent positive control for immunofluorescence studies . Confocal microscopy with Z-stack acquisition is recommended for precise subcellular localization analysis of SPATA19.

How does SPATA19 expression correlate with male fertility parameters in research models?

Current research suggests a significant correlation between SPATA19 expression levels and male fertility parameters. SPATA19 functions in spermiogenesis and is specifically expressed in testis tissue . Studies examining SPATA19 expression across different stages of spermatogenesis indicate its crucial role during the elongation phase of spermatids. The protein's localization to the mitochondrial outer membrane suggests involvement in mitochondrial sheath formation around the sperm flagellum, which is essential for sperm motility and energy production .

In research models with reduced SPATA19 expression, investigators have observed altered sperm morphology, particularly in the midpiece region where mitochondria form the mitochondrial sheath. Quantitative analysis of sperm parameters in these models shows:

These correlations suggest that SPATA19 antibodies can serve as valuable tools for investigating male infertility cases where mitochondrial function in sperm may be compromised. When designing such studies, it is advisable to incorporate multiple fertility parameters and correlate them with SPATA19 expression levels using quantitative immunostaining or Western blot analysis .

What techniques can be used to study SPATA19 interactions with mitochondrial proteins?

Investigating SPATA19 interactions with mitochondrial proteins requires sophisticated molecular techniques that maintain protein-protein interactions while providing specific detection. The following methodological approaches are recommended:

• Co-immunoprecipitation (Co-IP): Using SPATA19 antibodies to pull down protein complexes followed by mass spectrometry analysis can identify novel interaction partners . This approach requires:

  • Gentle lysis conditions using buffers containing 1% NP-40 or 0.5% Triton X-100

  • Pre-clearing lysates with protein A/G beads

  • Overnight incubation with SPATA19 antibody at 4°C

  • SDS-PAGE and immunoblotting for suspected interaction partners

• Proximity Ligation Assay (PLA): This technique allows visualization of protein interactions in situ with high specificity by detecting proteins that are within 40 nm of each other:

  • Fix tissues or cells as for immunofluorescence

  • Incubate with primary antibodies against SPATA19 and suspected interaction partners

  • Apply PLA probes and perform ligation and amplification

  • Analyze using fluorescence microscopy

• FRET (Fluorescence Resonance Energy Transfer): For studying dynamic interactions in live cells:

  • Generate fluorescent protein-tagged constructs of SPATA19 and potential partners

  • Transfect into appropriate cell lines

  • Measure energy transfer between fluorophores using spectral imaging

When studying SPATA19's mitochondrial interactions, include controls for mitochondrial outer membrane proteins such as TOMM20 or VDAC, which can serve as positive controls for localization and potential interaction studies .

How can researchers address cross-reactivity concerns when using SPATA19 antibodies in multi-protein analysis?

Cross-reactivity remains a significant challenge when using antibodies for multi-protein analysis, particularly with SPATA19 antibodies. To address these concerns, researchers should implement a systematic validation approach:

• Epitope analysis: Review the immunogen sequence used to generate the SPATA19 antibody and perform in silico analysis to identify potential cross-reactive proteins. The fusion protein immunogens used for commercial SPATA19 antibodies should be assessed for unique epitopes .

• Knockout/knockdown validation: Use SPATA19 knockout or knockdown models to confirm antibody specificity. A true SPATA19-specific antibody should show significantly reduced or absent signal in these models.

• Peptide competition assays: Pre-incubate the SPATA19 antibody with excess immunizing peptide before application to samples. Specific signals should be blocked by this treatment.

• Multiple antibody approach: Use two or more SPATA19 antibodies raised against different epitopes. Concordant results strengthen specificity claims.

• Western blot specificity assessment: In addition to the expected 19 kDa band, document any additional bands that might represent cross-reactive proteins or SPATA19 isoforms .

For multiplex studies combining SPATA19 with other antibodies, consider:

These methodical approaches help ensure that observed signals genuinely represent SPATA19 rather than cross-reactive proteins, particularly important when studying testicular tissue with its complex protein expression patterns .

What are common challenges in SPATA19 antibody applications and how can they be resolved?

Researchers frequently encounter several challenges when working with SPATA19 antibodies. The following table outlines common problems and their methodological solutions:

For technical inconsistencies, implement rigorous quality control measures including:

• Regular antibody validation using positive controls (testis tissue)

• Lot-to-lot testing when receiving new antibody batches

• Storage at recommended conditions (-20°C)

• Avoiding repeated freeze-thaw cycles

These systematic approaches can significantly improve reproducibility and reliability in SPATA19 detection across different applications.

How can researchers validate the specificity of SPATA19 antibodies in their experimental systems?

Validating antibody specificity is crucial for ensuring reliable research outcomes, particularly for proteins like SPATA19 with tissue-specific expression patterns. A comprehensive validation strategy includes:

For each new experimental system or critical study, researchers should implement at least three of these validation approaches to establish confidence in their SPATA19 antibody's specificity before proceeding with full experimental datasets.

How might SPATA19 antibodies contribute to understanding male infertility mechanisms?

SPATA19 antibodies represent valuable tools for investigating unexplored aspects of male infertility, particularly cases involving mitochondrial dysfunction in sperm. Future research applications could focus on:

• Clinical correlations: Using SPATA19 antibodies to assess protein expression in testicular biopsies from infertile men compared to fertile controls. This approach could identify a subset of infertility cases associated with SPATA19 dysregulation in the mitochondrial sheath formation .

• Mechanistic studies: Investigating the temporal relationship between SPATA19 expression and mitochondrial sheath assembly during spermiogenesis. Dual immunolabeling with SPATA19 antibodies and mitochondrial markers across developmental stages could reveal critical checkpoints in this process .

• Post-translational modifications: Using modification-specific antibodies alongside general SPATA19 antibodies to determine how phosphorylation, ubiquitination, or other modifications affect SPATA19 function in sperm development.

• Therapeutic development: Screening for compounds that modulate SPATA19 expression or function, potentially opening avenues for treating specific forms of male infertility.

• Environmental impact assessment: Utilizing SPATA19 antibodies to evaluate how environmental toxicants affect spermatogenesis at the molecular level, with SPATA19 serving as a sensitive marker for mitochondrial integrity in sperm.

These research directions could significantly advance our understanding of the molecular basis of male infertility, potentially leading to new diagnostic or therapeutic approaches for previously unexplained cases .

What emerging techniques could enhance SPATA19 protein interaction studies in reproductive biology?

Emerging technologies offer exciting possibilities for more comprehensive characterization of SPATA19 protein interactions in reproductive biology:

• Proximity-dependent biotinylation (BioID or TurboID): By fusing SPATA19 to a biotin ligase, researchers can identify proteins in close proximity to SPATA19 in living cells. This approach could reveal transient interactions missed by traditional co-immunoprecipitation:

  • Generate SPATA19-BioID fusion constructs

  • Express in relevant cell lines or testicular organ cultures

  • Purify biotinylated proteins and identify by mass spectrometry

  • Validate interactions using SPATA19 antibodies in orthogonal assays

• Single-cell proteomics: Combining single-cell isolation techniques with highly sensitive protein detection methods:

  • Isolate individual spermatogenic cells at different developmental stages

  • Analyze SPATA19 expression and interaction partners

  • Correlate with developmental state and functional parameters

• Super-resolution microscopy: Techniques such as STORM or PALM offer nanoscale resolution of protein localization:

  • Use fluorophore-conjugated SPATA19 antibodies

  • Visualize precise mitochondrial localization beyond diffraction limits

  • Perform multi-color imaging to map SPATA19 relative to other mitochondrial proteins

• Cryo-electron tomography: This technique could visualize the integration of SPATA19 into the mitochondrial outer membrane at molecular resolution, potentially revealing structural mechanisms of function.

• CRISPR-based screening: Combining CRISPR-Cas9 genome editing with SPATA19 antibody-based phenotyping could identify genetic modifiers of SPATA19 function:

  • Create CRISPR libraries targeting mitochondrial genes

  • Screen for altered SPATA19 localization or function

  • Validate hits using SPATA19 antibodies in detailed follow-up studies

These cutting-edge approaches, when coupled with well-validated SPATA19 antibodies, promise to significantly advance our understanding of this protein's role in reproductive biology and potentially expand its known functions beyond current knowledge .

What are the key publications for researchers studying SPATA19?

While the search results include limited information on published SPATA19 research, they indicate there are at least 16 PubMed citations related to SPATA19 and specifically mention that the Proteintech SPATA19 antibody (16656-1-AP) has been cited in at least 2 publications for Western blot applications and 2 publications for immunofluorescence applications . Researchers are encouraged to explore these publications for detailed methodologies and findings.

For comprehensive literature review, researchers should search databases using all known synonyms for SPATA19, including:

• SPATA19

• Spergen-1

• spergen1

• SPAS1

• CT132

• FLJ25851

The gene has been assigned the NCBI Gene ID 219938 , which can also be used for comprehensive literature searches across multiple databases.

How should researchers properly store and handle SPATA19 antibodies to maintain optimal performance?

Proper storage and handling of SPATA19 antibodies is crucial for maintaining their performance over time. Based on manufacturer recommendations: