SPATS1 Antibody

What is SPATS1 protein and why is it of interest to researchers?

SPATS1 (spermatogenesis-associated, serine-rich 1) is an evolutionarily conserved, testis-specific protein that is differentially expressed during male meiotic prophase. The protein was first detected in embryo testes at 17.5-18 days post-coitum (dpc) and reaches maximum expression levels at 21 days postpartum (dpp), coinciding with the pachytene stage of meiosis . SPATS1 is of particular research interest because some studies have suggested potential links between SPATS1 underexpression/mutation and human pathologies such as male infertility and testicular cancer . SPATS1 is highly conserved across metazoans, suggesting an important evolutionary role, though recent knockout studies have revealed it is not individually essential for mouse fertility .

What types of SPATS1 antibodies are available for research applications?

Researchers have access to several types of SPATS1 antibodies, including polyclonal antibodies such as the Rabbit Polyclonal Anti-SPATS1 Antibody, which targets human SPATS1 at a concentration of 0.4 mg/ml . These antibodies are typically validated for multiple applications including immunohistochemistry (IHC), immunocytochemistry-immunofluorescence (ICC-IF), and Western blotting (WB) . Additionally, antibodies used in ELISA kits are available for the quantitative detection of SPATS1 in various sample types .

What sample types can be analyzed using SPATS1 antibodies?

SPATS1 antibodies can be used to analyze various biological samples including cell culture supernatants, serum, plasma, and other biological fluids . In research contexts, these antibodies have been used to detect SPATS1 in tissue sections (particularly testicular tissue), cell lysates, and protein extracts. The selection of appropriate sample preparation methods depends on the specific application (IHC, Western blot, or ELISA) and should be optimized based on the antibody manufacturer's recommendations .

How should I design a Western blot experiment to detect SPATS1 protein?

When designing Western blot experiments for SPATS1 detection, follow these methodological steps for optimal results:

• Determine linear range: First establish the linear range for your specific SPATS1 antibody by performing serial 1:2 dilutions of your sample in Laemmli buffer .

• Sample loading design: Counterbalance sample positions across the gel by treatment condition to minimize position-based artifacts. Load approximately 15 μg of total protein per sample in separate lanes on a precast 10-20% Tris-HCl polyacrylamide gel .

• Electrophoresis conditions: Run the gel at 200V for approximately 30 minutes in SDS buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3) .

• Membrane transfer: Transfer proteins to a nitrocellulose membrane in cold transfer buffer (25 mM Tris, 192 mM glycine, 20% ethanol, pH 8.3). Confirm transfer using Ponceau S stain followed by blocking in appropriate blocking buffer .

• Antibody incubation: Block the membrane for 1 hour in blocking buffer containing 0.1% Tween-20, then incubate overnight with primary SPATS1 antibody at 4°C (typically at 1:1000-1:2000 dilution) .

• Detection: After washing, incubate with appropriate secondary antibody (such as IRdye-labeled goat anti-rabbit at 1:30,000) for 1 hour, then wash thoroughly before imaging .

• Technical replicates: For rigorous results, run at least three independent replications and use appropriate loading controls like beta-actin for covariance correction .

What are the key considerations for using SPATS1 ELISA kits in research?

When utilizing SPATS1 ELISA kits for quantitative analysis, consider the following methodological aspects:

• Assay principle: SPATS1 ELISA kits typically employ a two-site sandwich ELISA methodology. The microplate is pre-coated with an antibody specific for SPATS1, and samples are added so that any SPATS1 present binds to the immobilized antibody .

• Procedure overview: After sample binding, a biotin-conjugated SPATS1-specific antibody is added, followed by Streptavidin-HRP. A substrate solution creates color development proportional to SPATS1 concentration, which is measured colorimetrically .

• Sample preparation: Ensure proper sample preparation—SPATS1 can be measured in cell culture supernatants, plasma, serum, and other biological fluids. Each sample type may require specific preparation protocols .

• Duration consideration: The assay involves multiple steps with a working time of approximately 3-5 hours, depending on operator experience .

• Specificity: Select kits with validated specificity showing no significant cross-reactivity or interference between human SPATS1 and analogues .

• Controls: Always include appropriate standards and controls to establish a reliable standard curve for accurate quantification .

How can I validate the specificity of my SPATS1 antibody?

Validating SPATS1 antibody specificity is crucial for experimental reliability. Implement these methodological approaches:

• Positive and negative controls: Use tissue samples known to express SPATS1 (such as testicular tissue) as positive controls, and non-expressing tissues as negative controls. Based on current research, SPATS1 is primarily expressed in testicular tissues, with highest levels in primary spermatocytes .

• Knockout validation: The gold standard for antibody validation is testing in SPATS1 knockout models. Researchers have generated Spats1 loss-of-function mouse models using CRISPR/Cas9 technology which can serve as excellent negative controls .

• Multiple detection methods: Validate antibody specificity across multiple platforms (Western blot, IHC, ICC-IF) to ensure consistent detection patterns .

• Peptide competition: Perform peptide competition assays where pre-incubation of the antibody with its immunizing peptide should abolish specific signal.

• Alternative antibodies: When possible, compare results using alternative antibodies targeting different epitopes of SPATS1 to confirm detection pattern consistency .

How do I investigate potential correlations between SPATS1 expression and male fertility parameters?

To investigate correlations between SPATS1 expression and fertility parameters, consider this research methodology:

• Sample collection: Obtain appropriate samples from subjects with varying fertility statuses. Previous research has examined SPATS1 expression in men with severely impaired spermatogenesis compared to controls .

• Expression analysis methods:

  • Quantitative RT-PCR for SPATS1 mRNA expression

  • Western blot for protein expression using validated SPATS1 antibodies

  • Immunohistochemistry on testicular biopsies to analyze cellular localization patterns

• Correlation with fertility parameters: Analyze SPATS1 expression in relation to:

  • Sperm concentration and motility parameters

  • Spermatogenesis stages using histological analysis

  • Hormonal profiles (testosterone, FSH, LH)

• Statistical approach: Implement appropriate statistical methods to establish correlations:

  • Use linear mixed models to account for technical and biological variability

  • Apply covariance correction with established housekeeping genes/proteins

  • Run multiple replications with appropriate power calculations

• Comparative analysis: Although Spats1 knockout mice showed no overt fertility phenotype , human studies suggest decreased expression in men with impaired spermatogenesis, indicating possible species-specific differences that warrant careful comparative analysis.

What experimental approaches would help resolve contradictory findings regarding SPATS1's role in fertility?

Current research presents an interesting contradiction: while some human studies suggest associations between SPATS1 underexpression and fertility issues, knockout mouse models show no fertility phenotype . To address this contradiction:

• Species-specific investigations:

  • Compare SPATS1 expression patterns across species (human, mouse, rat, bovine)

  • Analyze protein interaction partners that might differ between species

  • Investigate compensatory mechanisms that might exist in mice but not humans

• Multifactorial analysis:

  • Investigate SPATS1 in combination with other fertility-related genes

  • Examine environmental factors that might interact with SPATS1 function

  • Study SPATS1 in various genetic backgrounds to identify potential modifier genes

• Functional genomics approaches:

  • Perform RNA-seq analysis on tissues from wild-type and knockout models

  • Conduct proteomics studies to identify changes in protein expression patterns

  • Investigate post-translational modifications of SPATS1, as it is known to be highly phosphorylatable

• Environmental and physiological stress models:

  • Test fertility of knockout models under various stress conditions

  • Investigate age-dependent effects (studies show different SPATS1 requirements in young versus aged animals)

• Clinical correlations:

  • Design larger cohort studies in humans with fertility issues

  • Perform exome sequencing to identify SPATS1 mutations in infertile populations

  • Correlate with specific clinical parameters of infertility

How can I investigate potential connections between SPATS1 and testicular cancer?

To investigate potential connections between SPATS1 and testicular cancer:

• Expression analysis in cancer samples:

  • Compare SPATS1 expression levels between normal testicular tissue and seminoma samples using validated antibodies

  • Perform IHC to examine cellular localization patterns in cancerous versus normal tissue

  • Conduct quantitative protein analysis using Western blot or ELISA

• Genetic analysis approaches:

  • Screen for SPATS1 mutations in testicular cancer samples, particularly seminomas, as exome-wide sequencing studies have suggested possible associations

  • Perform targeted sequencing of SPATS1 in large cohorts of testicular cancer patients

  • Analyze copy number variations that might affect SPATS1 expression

• Functional studies:

  • Develop cell culture models with SPATS1 knockdown/overexpression in testicular cell lines

  • Assess changes in proliferation, migration, and invasion capacity

  • Investigate potential involvement in the Wnt signaling pathway, which has been suggested in previous research and is often dysregulated in cancer

• Animal models:

  • While basic Spats1 knockout mice showed no spontaneous tumor development , consider creating compound mutant models with known testicular cancer predisposition genes

  • Analyze tumor incidence, progression, and metastasis in these models

• Integration of multi-omics data:

  • Correlate SPATS1 expression/mutation data with broader genomic, transcriptomic, and proteomic profiles of testicular cancers

  • Identify potential biomarker signatures that include SPATS1

What are the common issues when using SPATS1 antibodies in Western blot experiments and how can they be resolved?

When working with SPATS1 antibodies in Western blot experiments, researchers may encounter these common issues:

• Weak or no signal:

  • Solution: Optimize antibody concentration through titration experiments

  • Solution: Increase protein loading (up to 30μg) while ensuring you remain in the linear range

  • Solution: Extend primary antibody incubation time to overnight at 4°C

  • Solution: Verify sample preparation method preserves SPATS1 integrity (avoid excessive freeze-thaw cycles)

• High background:

  • Solution: Increase blocking time and washing steps

  • Solution: Dilute primary antibody further (test range from 1:500 to 1:5000)

  • Solution: Use fresh blocking buffer with increased Tween-20 concentration (up to 0.2%)

  • Solution: Ensure membrane is properly blocked before antibody incubation

• Multiple bands or unexpected band size:

  • Solution: Verify antibody specificity using knockout controls if available

  • Solution: Consider post-translational modifications, as SPATS1 is highly phosphorylatable

  • Solution: Adjust gel percentage to better resolve proteins in the expected molecular weight range

  • Solution: Consider alternative antibodies targeting different epitopes of SPATS1

• Inconsistent results between replicates:

  • Solution: Implement technical triplicates as recommended for Western blot reproducibility

  • Solution: Counterbalance sample positions across the gel by treatment condition

  • Solution: Use appropriate loading controls and statistical analysis as covariates

  • Solution: Standardize all steps of the protocol including sample preparation, protein quantification, and loading

How can I optimize SPATS1 immunohistochemistry for different tissue types?

To optimize SPATS1 immunohistochemistry across various tissue types:

• Fixation optimization:

  • For testicular tissue (primary SPATS1 expression site), optimize fixation time (typically 24-48 hours in 10% neutral buffered formalin)

  • Consider alternative fixatives such as Bouin's solution for better morphological preservation of testicular tissues

  • For frozen sections, fixation in cold acetone or 4% paraformaldehyde can preserve antigenicity

• Antigen retrieval methods:

  • Compare heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) versus EDTA buffer (pH 9.0)

  • Optimize retrieval time (typically 10-30 minutes) and method (microwave, pressure cooker, or water bath)

  • For testicular tissue, gentler retrieval methods may be preferred to preserve morphology

• Blocking and antibody conditions:

  • Test various blocking solutions (BSA, normal serum, commercial blockers)

  • Determine optimal primary antibody dilution (typically starting at 1:100-1:500 for IHC)

  • Optimize incubation conditions (1 hour at room temperature versus overnight at 4°C)

• Detection system selection:

  • Compare different detection systems (ABC, polymer-based) for sensitivity and specificity

  • For dual staining to co-localize with other proteins, select compatible detection systems with distinct chromogens

  • Consider fluorescent detection for co-localization studies with other testicular markers

• Controls and validation:

  • Include tissue sections from SPATS1 knockout models as negative controls

  • Use testicular tissue sections at different developmental stages, as SPATS1 expression varies temporally (highest in pachytene stage spermatocytes)

  • Perform peptide competition assays to confirm specificity of staining pattern

What statistical approaches should I use when analyzing SPATS1 expression data across experimental groups?

For rigorous statistical analysis of SPATS1 expression data:

• Experimental design considerations:

  • Plan for adequate biological replicates (minimum n=3, preferably n≥5)

  • Include technical triplicates for Western blot experiments

  • Counterbalance sample positions across gels by treatment condition to minimize position effects

• Appropriate statistical models:

  • For simple comparisons between two groups, use t-tests with Welch's correction for unequal variances if necessary

  • For multiple groups, employ ANOVA followed by appropriate post-hoc tests (Tukey, Bonferroni)

  • For complex designs, consider linear mixed models that can account for both fixed and random effects

• Covariate incorporation:

  • Use loading controls (e.g., beta-actin) as covariates rather than simple normalization factors

  • Treat technical replication statistically as a random factor in the analysis

  • Consider appropriate housekeeping genes/proteins based on your experimental system

• Effect size and power analysis:

  • Report standardized regression coefficient effect sizes rather than just p-values

  • Calculate observed statistical power through Monte Carlo simulation (1000x) run on the fitted model

  • Consider power calculations for future experiments based on observed effect sizes

• Reporting standards:

  • Set significance threshold (typically p<0.05) before beginning analysis

  • Present data with appropriate visualization (box plots, scatter plots with mean ± SEM)

  • Report both effect sizes and p-values for comprehensive interpretation

What emerging research questions about SPATS1 should investigators consider exploring?

Based on current knowledge gaps and contradictory findings, several promising research directions emerge:

• Molecular function characterization:

  • Investigate the molecular function of SPATS1 through protein interaction studies

  • Explore its reported connections to the Wnt signaling pathway and determine if this relationship is tissue-specific

  • Characterize the significance of its high phosphorylation potential and identify key phosphorylation sites

• Species-specific roles:

  • Examine why SPATS1 appears associated with fertility in humans but is dispensable in mice

  • Compare SPATS1 interaction networks across species to identify potential compensatory mechanisms

  • Investigate potential redundancy with other spermatogenesis-associated proteins

• Clinical associations:

  • Conduct larger clinical studies on SPATS1 expression in various male fertility disorders

  • Explore potential associations with specific types of testicular cancers beyond seminomas

  • Investigate whether SPATS1 could serve as a biomarker for specific subtypes of male infertility

• Regulatory mechanisms:

  • Study the transcriptional and post-transcriptional regulation of SPATS1

  • Investigate epigenetic mechanisms controlling its testis-specific expression

  • Examine how environmental factors might influence SPATS1 expression and function

• Therapeutic implications:

  • Explore whether modulation of SPATS1 could have therapeutic applications in certain forms of male infertility

  • Investigate potential as a target in testicular cancer cases with SPATS1 mutations