spata18 Antibody

What is SPATA18 and what are its primary biological functions?

SPATA18 is a multifunctional protein initially identified as a cytoplasmic protein in elongate spermatids that plays a role in the differentiation process of elongate spermatids to mature spermatozoa . More recent research has established SPATA18 as a p53-inducible protein with critical functions in mitochondrial quality control . It induces lysosome-like organelles within mitochondria that eliminate oxidized mitochondrial proteins, effectively functioning as a tumor suppressor through its role in maintaining mitochondrial integrity .

The protein exists in at least three isoforms, with most commercially available antibodies detecting the two longest isoforms . SPATA18's functions span reproductive biology and cellular maintenance, making it a target of interest across multiple research fields including cancer biology, reproductive sciences, and studies of mitochondrial dynamics.

What applications are most suitable for SPATA18 antibodies?

SPATA18 antibodies are compatible with multiple experimental applications, with the most common and validated being:

• Western Blotting (WB): Effective for detecting SPATA18 protein expression levels and confirming specificity with appropriate controls

• Enzyme-Linked Immunosorbent Assay (ELISA): Useful for quantitative detection of SPATA18

• Immunohistochemistry (IHC): Valuable for localizing SPATA18 in tissue sections and evaluating expression patterns in normal versus pathological samples

When selecting an application, researchers should consider that validation data exists primarily for these three techniques. For Western blotting specifically, published results have demonstrated successful detection of SPATA18 in rat lung tissue lysates at concentrations of approximately 1 μg/mL .

How do I select the appropriate SPATA18 antibody for cross-species reactivity?

When planning experiments involving multiple species, consider these validated cross-reactivity profiles:

For maximum versatility, rabbit-derived polyclonal antibodies typically offer broader cross-reactivity. When working with less common research species (cow, horse), verify reactivity claims with validation data. Always perform a literature review to identify which antibodies have been successfully used in published studies with your species of interest .

What controls should I include when using SPATA18 antibodies?

For rigorous experimental design with SPATA18 antibodies, implement these essential controls:

• Blocking peptide control: Run parallel experiments with and without the blocking peptide to confirm antibody specificity, as demonstrated in rat lung tissue lysate experiments

• Positive control: Include samples known to express SPATA18 (lung tissue is recommended based on successful detection in published work)

• Negative control: Include primary antibody omission controls and tissues/cells where SPATA18 expression is expected to be minimal

• Loading control: Use appropriate housekeeping proteins (β-actin, GAPDH) for Western blots to normalize SPATA18 signals across samples

The validation data provided with SPATA18 antibodies typically includes Western blot analysis showing clear differentiation between specific signal and blocked controls, confirming antibody specificity .

How can I optimize immunohistochemical detection of SPATA18 in cancer tissues?

For optimal immunohistochemical detection of SPATA18 in cancer tissues, implement this methodological approach:

• Tissue preparation: Fix tissues in 10% neutral buffered formalin and embed in paraffin. Section at 3-5 μm thickness .

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 15-20 minutes .

• Blocking and antibody application:

  • Block with TBS solution containing 1% BSA

  • Apply primary SPATA18 antibody (optimal dilution range: 1:100 to 1:200)

  • Incubate overnight at 4°C

  • Apply appropriate secondary antibody conjugated with Streptomyces avidin-peroxidase

  • Incubate for 1 hour at room temperature

• Scoring system implementation: For quantitative assessment, use a combined scoring system:

  • Staining intensity (0-3): no staining (0), weak (1), moderate (2), strong (3)

  • Proportion of positive cells (0-3): percentage-based scoring

  • Calculate final score by summing intensity and proportion scores

This methodology has been validated in studies of colorectal cancer and clear cell renal cell carcinoma, demonstrating that SPATA18 expression correlates with clinical outcomes and can serve as a prognostic biomarker .

What are the critical considerations when investigating SPATA18's relationship with p53 pathway activity?

When investigating the relationship between SPATA18 and the p53 pathway, researchers should address several critical considerations:

• Regulatory complexity: While SPATA18 is reported to be p53-inducible, its expression is also regulated by other mechanisms including promoter methylation. Research has shown that SPATA18 promoter methylation occurs in only approximately 9% of colorectal cancer cases, suggesting that additional regulatory mechanisms are important .

• P53 mutation heterogeneity: The relationship between p53 mutations and SPATA18 expression is complex. Studies in colorectal cancer have found no significant correlation between SPATA18 expression and p53 immunoreactivity (a surrogate marker for TP53 mutation) . This may be due to the diversity of p53 mutant forms with variable transcriptional activities.

• Methodological approach:

  • Assess both SPATA18 expression and p53 status simultaneously in the same samples

  • Employ multiple techniques beyond immunohistochemistry, such as sequencing TP53 to identify specific mutations

  • Consider evaluating additional p53 pathway components to provide context for SPATA18 regulation

• Functional validation: Design experiments that manipulate p53 activity (activation with nutlin-3a or suppression with siRNA) and measure effects on SPATA18 expression to establish causality rather than correlation.

Understanding this complex relationship requires integrating data on p53 mutation status, SPATA18 expression patterns, and functional outcomes in specific tissue contexts .

How does SPATA18 expression correlate with tumor characteristics and patient prognosis?

SPATA18 expression demonstrates significant correlations with tumor characteristics and patient outcomes, as evidenced by comprehensive studies in multiple cancer types:

These findings suggest SPATA18 functions as a tumor suppressor, with its decreased expression associated with more aggressive disease features and poorer prognosis. When designing studies to investigate SPATA18 as a prognostic biomarker, researchers should implement multivariate analyses that control for established prognostic factors to determine its independent contribution to outcome prediction .

What are the recommended methodologies for investigating SPATA18's role in mitochondrial quality control?

To effectively investigate SPATA18's role in mitochondrial quality control, implement these methodological approaches:

• Mitochondrial morphology and function assessment:

  • Fluorescence microscopy using MitoTracker dyes to visualize mitochondrial networks

  • Transmission electron microscopy to identify SPATA18-induced lysosome-like structures within mitochondria

  • Mitochondrial membrane potential measurement using JC-1 or TMRM dyes

  • Oxygen consumption rate (OCR) analysis using Seahorse XF analyzers

• SPATA18 manipulation strategies:

  • Overexpression systems using tagged SPATA18 constructs to track localization

  • RNA interference (siRNA/shRNA) or CRISPR-Cas9 to create SPATA18 knockdown/knockout models

  • Inducible expression systems to control timing of SPATA18 expression

• Mitochondrial protein quality assessment:

  • Measurement of oxidized protein levels using OxyBlot methodology

  • Co-immunoprecipitation to identify SPATA18 interaction partners

  • Proximity labeling techniques (BioID, APEX) to map the SPATA18 interactome within mitochondria

• Functional readouts:

  • Mitophagy flux assays using mt-Keima or mito-QC reporter systems

  • ROS measurement using CM-H2DCFDA or MitoSOX

  • ATP production quantification

These methodologies align with current understanding of SPATA18 as a mediator of mitochondrial quality control that induces lysosome-like organelles to eliminate oxidized mitochondrial proteins, thereby maintaining mitochondrial integrity and function .

What are the optimal storage and handling conditions for SPATA18 antibodies?

For maximum stability and performance of SPATA18 antibodies, adhere to these storage and handling guidelines:

• Long-term storage: Store antibodies at -20°C, where they remain stable for approximately one year . Avoid storing antibodies at temperatures above recommended ranges, as this can lead to accelerated degradation.

• Handling precautions:

  • Minimize freeze-thaw cycles, as repeated freezing and thawing can diminish antibody activity and specificity

  • Consider aliquoting stock antibody solutions to minimize freeze-thaw cycles

  • When working with antibodies, maintain sterile technique to prevent microbial contamination

• Working dilutions: Prepare working dilutions immediately before use and avoid storing diluted antibody solutions for extended periods.

• Buffer compatibility: Most SPATA18 antibodies are supplied in PBS containing 0.02% sodium azide as a preservative . Be aware that sodium azide can inhibit peroxidase activity, which may affect certain detection methods.

Following these guidelines will help ensure consistent performance in experimental applications and maximize the usable lifespan of SPATA18 antibodies .

How can conflicting SPATA18 expression data between different detection methods be reconciled?

When faced with conflicting SPATA18 expression data across different detection platforms, implement this systematic troubleshooting approach:

• Methodological differences assessment:

  • Examine antibody epitope locations: Different antibodies may target distinct regions of SPATA18, potentially detecting different isoforms

  • Review detection sensitivity thresholds: Techniques vary in sensitivity (Western blot vs. immunohistochemistry vs. ELISA)

  • Consider sample preparation variations: Protein extraction methods may affect epitope availability

• Isoform-specific detection:

  • At least three isoforms of SPATA18 exist, with most antibodies detecting only the two longest forms

  • Verify which isoforms your specific antibody detects

  • Design primers or select antibodies that can discriminate between isoforms

• Validation approach:

  • Implement orthogonal techniques (e.g., validate protein expression findings with mRNA analysis)

  • Use multiple antibodies targeting different epitopes on SPATA18

  • Include appropriate positive and negative controls, including blocking peptide controls

• Data integration strategy:

  • Weight evidence based on methodological rigor

  • Consider biological context of each sample type

  • Develop a composite assessment that acknowledges technical limitations

This approach has been valuable in reconciling SPATA18 expression findings in cancer studies, where different detection methods (serum markers vs. tissue immunohistochemistry) revealed complementary rather than contradictory information about SPATA18's role .

What are the best practices for validating SPATA18 antibody specificity?

To rigorously validate SPATA18 antibody specificity, implement these comprehensive best practices:

• Blocking peptide competition assays:

  • Perform parallel Western blots with antibody pre-incubated with and without blocking peptide

  • Disappearance of signal in the presence of blocking peptide confirms specificity

  • Document results showing clear comparison as demonstrated in rat lung tissue lysate experiments

• Genetic validation strategies:

  • Test antibody in SPATA18 knockout/knockdown systems

  • Observe loss of signal in SPATA18-depleted samples

  • Complement with overexpression systems showing increased signal intensity

• Cross-reactivity assessment:

  • Test against related SPATA family members to confirm absence of cross-reactivity

  • Verify species specificity matches manufacturer claims

  • Examine tissues known to have variable SPATA18 expression

• Multiple epitope targeting:

  • Compare results using antibodies targeting different SPATA18 regions

  • Consistent results across different antibodies increase confidence in specificity

  • Document epitope locations relative to known SPATA18 domains

• Orthogonal method validation:

  • Correlate protein detection with mRNA expression data

  • Compare results across different application methods (WB, IHC, ELISA)

  • Analyze subcellular localization patterns for consistency with known biology

These validation steps are essential for generating reliable data, especially in cancer research where SPATA18 expression has prognostic significance .

What experimental design considerations are important when studying SPATA18 in cancer models?

When designing experiments to study SPATA18 in cancer models, incorporate these critical design considerations:

• Sample selection and characterization:

  • Include matched tumor and adjacent normal tissue when possible

  • Comprehensively characterize samples for relevant clinical and pathological parameters

  • Consider stratification by tumor grade, stage, and molecular subtype

• Expression analysis optimization:

  • Implement standardized scoring systems combining staining intensity and proportion

  • Use receiver operating characteristic (ROC) curve analysis to determine optimal cutoff values for defining "high" versus "low" expression

  • Document AUC values with confidence intervals (example: AUC of 0.595; 95% CI = 0.50–0.69 for SPATA18 in colorectal cancer)

• Functional correlation studies:

  • Assess relationship between SPATA18 expression and proliferation markers (e.g., PHH3)

  • Investigate potential associations with p53 status and mitochondrial parameters

  • Examine correlation with established prognostic markers

• Survival analysis methodology:

  • Conduct both univariate (Kaplan-Meier) and multivariate (Cox regression) analyses

  • Stratify by relevant clinical parameters (T classification, clinical stage, metastasis status)

  • Report hazard ratios with confidence intervals

• Mechanistic investigation:

  • Design in vitro experiments using appropriate cell lines that recapitulate expression patterns seen in tumors

  • Consider pathway analysis using techniques like GSEA to identify associated biological processes

  • Investigate related pathways identified in ccRCC (B cell receptors, WNT targets, extracellular matrix, oxidative phosphorylation)

These design considerations have been successfully implemented in studies demonstrating SPATA18's prognostic significance in colorectal cancer and clear cell renal cell carcinoma .

How can SPATA18 antibodies be utilized to investigate the relationship between mitochondrial dysfunction and cancer progression?

SPATA18 antibodies offer valuable tools for exploring the intersection of mitochondrial dysfunction and cancer progression through these methodological approaches:

• Dual immunofluorescence/immunohistochemistry protocols:

  • Co-stain for SPATA18 and mitochondrial markers (TOMM20, COX IV)

  • Quantify co-localization in normal versus malignant tissues

  • Correlate mitochondrial SPATA18 localization with cancer stage and grade

• Gradient analysis across tumor progression:

  • Examine SPATA18 expression in tissue microarrays containing:

    • Normal tissue

    • Precancerous lesions

    • Early-stage cancer

    • Advanced/metastatic disease

  • Quantify changes in expression and localization along the progression continuum

• Functional mitochondrial assessments:

  • Correlate SPATA18 expression with mitochondrial membrane potential

  • Measure markers of oxidative damage in tissues with variable SPATA18 expression

  • Assess mitophagic flux in relation to SPATA18 levels

• Therapeutic response correlation:

  • Examine whether SPATA18 expression predicts response to therapies targeting mitochondrial function

  • Investigate if SPATA18 levels change following treatment with standard chemotherapeutics

This integrated approach leverages SPATA18's established role in mitochondrial quality control and its prognostic significance in cancers, providing mechanistic insights into how mitochondrial dysfunction contributes to cancer progression .

What methodological approaches can resolve contradictory findings regarding SPATA18's relationship with p53 in different tumor types?

To address contradictory findings regarding SPATA18's relationship with p53 across different tumor types, implement these methodological approaches:

• Comprehensive p53 characterization:

  • Sequence TP53 to identify specific mutations rather than relying solely on p53 immunoreactivity

  • Classify mutations based on functional impact (loss of function, gain of function, dominant negative)

  • Assess p53 target gene expression as a measure of pathway activity

• Tissue-specific regulatory network analysis:

  • Perform ChIP-seq to map p53 binding to the SPATA18 promoter in different tissue contexts

  • Assess epigenetic modifications (methylation, histone marks) at the SPATA18 promoter

  • Identify tissue-specific transcription factors that may cooperate with or antagonize p53

• Pathway integration approach:

  • Examine SPATA18 expression in relation to both p53 and p63 status

  • Investigate the role of promoter methylation in regulating SPATA18 expression independent of p53

  • Consider the impact of MDM2 amplification and other p53 pathway alterations

• Functional validation experiments:

  • Restore wild-type p53 in p53-mutant cell lines and assess SPATA18 induction

  • Introduce specific p53 mutants to determine their differential effects on SPATA18 expression

  • Manipulate SPATA18 directly to assess its contribution to p53-dependent and p53-independent phenotypes

This multifaceted approach addresses the complexity observed in colorectal cancer, where no significant correlation was found between SPATA18 expression and p53 immunoreactivity despite SPATA18 being a reported p53 target gene .

How can SPATA18 antibodies contribute to understanding the role of mitochondrial quality control in treatment resistance?

SPATA18 antibodies can be strategically employed to investigate mitochondrial quality control in treatment resistance through these methodological approaches:

• Pre- and post-treatment analysis protocol:

  • Collect paired samples before and after therapy

  • Quantify changes in SPATA18 expression and localization

  • Correlate expression patterns with treatment response and resistance development

• Cellular stress response characterization:

  • Expose cancer cell lines to therapeutic agents

  • Monitor SPATA18 induction and mitochondrial localization

  • Assess mitochondrial morphology and function in relation to SPATA18 dynamics

  • Correlate with markers of mitophagy and mitochondrial biogenesis

• Resistance model development:

  • Create resistant cell lines through chronic drug exposure

  • Compare SPATA18 expression and function between parental and resistant cells

  • Manipulate SPATA18 levels to determine impact on therapeutic sensitivity

• Clinical correlation strategy:

  • Develop a standardized IHC protocol for SPATA18 in clinical samples

  • Create a scoring system combining intensity and distribution

  • Analyze SPATA18 expression in responders versus non-responders

  • Integrate with markers of mitochondrial function and damage

This approach builds on findings that SPATA18 expression correlates with prognosis in multiple cancer types , suggesting it may play a role in treatment response pathways through its functions in maintaining mitochondrial integrity—a critical factor in cancer cell survival during therapy.

What is the optimal methodology for investigating SPATA18's role across different cancer types and stages?

To systematically investigate SPATA18's role across diverse cancer types and stages, implement this comprehensive methodological framework:

• Multi-cancer tissue microarray (TMA) analysis:

  • Design TMAs containing multiple cancer types with matched normal tissues

  • Standardize immunohistochemical protocols for consistent SPATA18 detection

  • Implement uniform scoring system combining intensity (0-3) and proportion (0-3) metrics

  • Analyze expression patterns stratified by cancer type, grade, and stage

• Integrated multi-omics approach:

  • Correlate protein expression (IHC) with mRNA levels (RNA-seq)

  • Assess promoter methylation status across cancer types

  • Conduct pathway analysis (GSEA) to identify cancer-specific associated pathways

  • Example pathways identified in ccRCC: B cell receptors, WNT targets, extracellular matrix, oxidative phosphorylation

• Functional validation protocol:

  • Select cell line panels representing multiple cancer types

  • Manipulate SPATA18 expression (overexpression/knockdown)

  • Assess impact on:

    • Proliferation and apoptosis

    • Migration and invasion

    • Mitochondrial function

    • Therapeutic response

• Clinical correlation framework:

  • Calculate diagnostic value using ROC curves

  • Perform survival analysis (Kaplan-Meier and Cox regression)

  • Stratify by cancer type and stage

  • Identify cancer types where SPATA18 has strongest prognostic value

This comprehensive methodology has been partially validated in studies of colorectal cancer and clear cell renal cell carcinoma, which demonstrated that high SPATA18 expression correlates with favorable prognosis, though with cancer-specific differences in the strength of association and pathway interactions .

What emerging applications of SPATA18 antibodies should researchers consider exploring?

Researchers should consider these emerging applications for SPATA18 antibodies that extend beyond current established uses:

• Liquid biopsy development:

  • Investigate SPATA18 detection in circulating tumor cells or exosomes

  • Develop protocols for capturing SPATA18-expressing cells from blood

  • Correlate with tissue expression patterns and clinical outcomes

• Therapeutic response monitoring:

  • Validate SPATA18 as a dynamic biomarker of treatment efficacy

  • Develop standardized protocols for tracking expression changes during therapy

  • Correlate with established response criteria and survival outcomes

• Mitochondrial pathology applications:

  • Expand SPATA18 antibody use to non-cancer mitochondrial disorders

  • Investigate expression in neurodegenerative conditions

  • Develop dual-staining protocols with markers of mitochondrial damage

• Single-cell analysis integration:

  • Adapt SPATA18 antibodies for mass cytometry (CyTOF) applications

  • Develop protocols for single-cell Western blotting

  • Integrate with spatial transcriptomics for tissue-context analysis

These emerging applications build upon SPATA18's established roles in mitochondrial quality control and cancer prognosis , expanding its utility as a research tool across multiple disciplines and technological platforms.

How might SPATA18 research evolve with advances in antibody and imaging technologies?

The future of SPATA18 research stands to be transformed by advances in antibody and imaging technologies through these developing methodologies:

• Super-resolution microscopy applications:

  • Implement STORM/PALM techniques to visualize SPATA18-mediated mitochondrial quality control at nanoscale resolution

  • Track individual SPATA18 molecules within mitochondrial subcompartments

  • Correlate spatial distribution with functional outcomes

• Multiplex imaging protocols:

  • Develop multiplexed immunofluorescence panels including SPATA18 and related mitochondrial markers

  • Implement cyclic immunofluorescence or imaging mass cytometry for comprehensive phenotyping

  • Create spatial maps of SPATA18 expression in relation to tumor microenvironment features

• Live-cell imaging advancements:

  • Generate knock-in fluorescent SPATA18 reporter cell lines

  • Employ optogenetic control of SPATA18 expression or localization

  • Monitor real-time dynamics of SPATA18-mediated mitochondrial quality control

• Next-generation antibody technologies:

  • Develop recombinant nanobodies against SPATA18 for improved tissue penetration

  • Create bifunctional antibodies linking SPATA18 to additional detection modalities

  • Engineer antibody fragments optimized for specific applications

These technological advances will enable researchers to move beyond static assessment of SPATA18 expression to dynamic, spatially-resolved understanding of its functions in mitochondrial quality control and its implications for cancer biology and treatment .

What standardization efforts would benefit the SPATA18 research community?

To advance SPATA18 research and improve cross-study comparability, the field would benefit from these standardization initiatives:

• Antibody validation standards:

  • Establish minimum validation criteria (specificity, sensitivity, reproducibility)

  • Create reference materials for antibody evaluation

  • Develop reporting guidelines for antibody characterization methods

  • Encourage publication of detailed protocols including blocking peptide controls

• Expression assessment harmonization:

  • Standardize scoring systems for immunohistochemistry combining:

    • Staining intensity (0-3 scale)

    • Proportion of positive cells (0-3 scale)

  • Establish consensus cutoffs for "high" versus "low" expression

  • Create digital pathology algorithms for automated, objective quantification

• Experimental design guidelines:

  • Define minimum requirements for control samples

  • Establish recommended cell line and tissue models

  • Create standard operating procedures for key SPATA18-related assays

  • Develop reporter constructs and genetic tools available to all researchers

• Data sharing infrastructure:

  • Build repositories for SPATA18 expression data across tissues and conditions

  • Create annotated image libraries of SPATA18 staining patterns

  • Establish platforms for sharing detailed methodological protocols