TMEM177 Antibody

What is TMEM177 and what cellular functions is it associated with?

TMEM177 (Transmembrane Protein 177) is a mitochondrial membrane protein that has been identified to associate with COX20, suggesting a potential role in mitochondrial function . While the full characterization of TMEM177 remains ongoing in the scientific community, research indicates it may participate in important cellular processes related to mitochondrial activity. The protein has been detected in various human tissues and cell lines, including HeLa, HepG2, and MCF-7 cells, suggesting widespread expression across different cell types . Understanding TMEM177's precise function requires targeted investigations using specific antibodies and molecular techniques.

What research applications is Anti-TMEM177 antibody typically used for?

Anti-TMEM177 antibody has been validated for multiple research applications including:

• Western blotting (WB) - For protein expression analysis and quantification

• Immunocytochemistry (ICC) - For cellular localization studies

• Immunohistochemistry on paraffin-embedded tissues (IHC-P) - For tissue expression pattern analysis

These applications enable researchers to investigate TMEM177 expression levels, subcellular localization, and tissue distribution patterns. The antibody's versatility makes it valuable for both basic characterization studies and more complex functional investigations of TMEM177 in various biological contexts.

What species reactivity does the TMEM177 antibody have?

Current commercially available monoclonal antibodies against TMEM177, such as those referenced in the literature, demonstrate specific reactivity to human TMEM177 . This species reactivity limitation should be considered when designing experiments, particularly those involving animal models. When studying TMEM177 in non-human systems, researchers should first verify cross-reactivity or seek species-specific antibodies. The human-specific nature of available antibodies makes them particularly suitable for studies using human cell lines, tissues, or clinical samples.

What is the typical subcellular localization of TMEM177?

Immunocytochemistry studies using Anti-TMEM177 antibody reveal predominantly cytoplasmic localization of TMEM177 in human cell lines . This cytoplasmic pattern is consistent with its reported association with mitochondria, as indicated by research on TMEM177's interaction with COX20, a mitochondrial protein . When performing localization studies, researchers should consider co-staining with mitochondrial markers to further elucidate the precise subcellular compartmentalization of TMEM177 within the cytoplasmic space.

What are the recommended dilutions for using TMEM177 antibody in various applications?

Based on validation studies, the following dilution ranges are recommended for optimal results with Anti-TMEM177 antibody:

These ranges serve as starting points and may require optimization based on your specific experimental conditions, sample types, and detection systems . It is advisable to perform initial titration experiments to determine the optimal antibody concentration for your particular application to maximize specific signal while minimizing background.

What fixation and permeabilization methods are optimal for TMEM177 immunocytochemistry?

For successful immunocytochemical detection of TMEM177, validation studies have established effective protocols using:

• Fixation: Paraformaldehyde fixation

• Permeabilization: 0.25% Triton X100 in PBS

This combination has been successfully applied in multiple cell lines including HeLa, HepG2, and MCF-7 cells. The fixation protocol preserves cellular architecture while maintaining TMEM177 antigenicity, while the permeabilization method ensures adequate antibody access to intracellular TMEM177. For optimal results, fix cells for 15-20 minutes at room temperature followed by permeabilization for 10 minutes before proceeding with antibody incubation.

How should TMEM177 antibody be stored to maintain activity?

To preserve antibody functionality and prevent degradation, the following storage conditions are recommended for Anti-TMEM177 antibody:

• Short-term storage (up to 1 month): +4°C after thawing

• Long-term storage: Aliquot and store at -20°C or -80°C

• Avoid repeated freeze/thaw cycles, which can significantly reduce antibody activity

When preparing working dilutions, it's advisable to use freshly thawed aliquots rather than repeatedly accessing the same stock. The antibody is typically supplied in a stabilizing formulation containing PBS (pH 7.4), 0.2% BSA, and 40% glycerol with 0.05% sodium azide as a preservative .

How does TMEM177 relate to mitochondrial function based on current research?

Current research indicates that TMEM177 is associated with mitochondrial processes, particularly through its interaction with COX20 . While the complete functional characterization is still evolving, this association suggests potential involvement in:

• Mitochondrial respiratory chain assembly or regulation

• Cellular energy metabolism

• Mitochondrial quality control mechanisms

Researchers investigating TMEM177's role in mitochondrial function should consider experimental approaches that monitor changes in mitochondrial morphology, membrane potential, respiratory capacity, and ATP production following TMEM177 manipulation. Colocalization studies with established mitochondrial markers and interaction analyses with known mitochondrial proteins would provide further insights into TMEM177's functional significance.

What experimental approaches are most effective for studying TMEM177's role in cellular pathways?

To thoroughly investigate TMEM177's functional roles, researchers should consider a multi-faceted experimental approach:

• Loss-of-function studies: siRNA knockdown or CRISPR-Cas9 gene editing to assess cellular consequences of TMEM177 depletion

• Gain-of-function studies: Overexpression systems to evaluate effects of increased TMEM177 expression

• Protein interaction studies: Co-immunoprecipitation or proximity labeling techniques to identify TMEM177 binding partners

• Live-cell imaging: Fluorescently tagged TMEM177 to monitor dynamic localization and trafficking

• Functional assays: Measurements of mitochondrial function in the context of TMEM177 manipulation

This comprehensive approach would provide complementary data points to establish TMEM177's biological significance and pathway associations.

What is known about tissue-specific expression patterns of TMEM177?

Immunohistochemical analyses have demonstrated TMEM177 expression in multiple human tissues. Validated tissues with confirmed TMEM177 expression include:

• Colon

• Breast

• Stomach

• Uterine muscle

This distribution suggests TMEM177 may have widespread expression across diverse tissue types. Understanding tissue-specific expression patterns provides valuable context for interpreting TMEM177's potential functional roles in different physiological systems. Researchers interested in tissue-specific functions should consider comparative expression analyses across normal tissues and their corresponding pathological states.

Why might I see multiple bands in my Western blot using TMEM177 antibody?

Multiple bands in Western blots using Anti-TMEM177 antibody may result from several factors:

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications can alter protein migration

• Protein isoforms: Alternative splicing may generate multiple TMEM177 variants

• Proteolytic degradation: Sample handling or incomplete protease inhibition may produce fragmentation

• Cross-reactivity: The antibody may detect structurally similar proteins, particularly at higher concentrations

To address this issue, researchers should:

• Include positive controls with verified TMEM177 expression

• Optimize sample preparation with comprehensive protease inhibitor cocktails

• Titrate antibody concentration to determine optimal specificity

• Consider using reducing and non-reducing conditions to evaluate potential influence of disulfide bonds

How can I reduce background staining in TMEM177 immunocytochemistry?

High background in immunocytochemistry experiments can obscure specific TMEM177 signals. To minimize background:

• Optimize blocking conditions: Extend blocking time (1-2 hours) and evaluate different blocking agents (BSA, normal serum, commercial blockers)

• Adjust antibody dilution: Test more dilute antibody solutions within the recommended range (1:100-1:200)

• Incorporate additional washing steps: Increase number and duration of washes between antibody incubations

• Evaluate secondary antibody specificity: Test secondary antibody alone to identify potential non-specific binding

• Optimize permeabilization: Excessive permeabilization can increase non-specific binding; titrate Triton X-100 concentration (0.1-0.25%)

These methodological refinements can significantly improve signal-to-noise ratio in TMEM177 immunocytochemistry experiments.

What are appropriate negative controls for TMEM177 antibody experiments?

Rigorous experimental design requires appropriate negative controls to validate TMEM177 antibody specificity:

• Isotype control: Use matched isotype (Mouse IgG1) at the same concentration as the TMEM177 antibody

• Secondary antibody only: Omit primary antibody to assess non-specific binding of detection system

• Blocking peptide competition: Pre-incubate antibody with excess TMEM177 recombinant protein to validate specificity

• TMEM177-knockout or knockdown samples: When available, analyze samples with confirmed TMEM177 depletion

• Tissues/cells known to be negative for TMEM177: Test antibody on samples without TMEM177 expression

Incorporating these controls enhances result interpretation and ensures scientific rigor in TMEM177 research.

How might TMEM177 research contribute to understanding mitochondrial disorders?

Given TMEM177's association with mitochondrial components like COX20 , investigations into this protein may provide valuable insights into mitochondrial disorders. Researchers could explore:

• TMEM177 expression patterns in patient samples with diagnosed mitochondrial diseases

• Functional consequences of TMEM177 mutations or expression changes in cellular models

• Potential role of TMEM177 in mitochondrial stress response mechanisms

• Therapeutic approaches targeting TMEM177 pathways for mitochondrial dysfunction

These research directions could potentially uncover novel mechanisms underlying mitochondrial pathologies and identify new diagnostic markers or therapeutic targets.

What methodological approaches should be considered for studying TMEM177 in disease models?

Researchers investigating TMEM177 in disease contexts should consider implementing:

• Patient-derived samples: Analysis of TMEM177 expression in relevant clinical specimens

• Disease-specific cell models: Generation of cell lines with disease-associated mutations affecting TMEM177 or its interaction partners

• Conditional knockout animal models: Tissue-specific or inducible TMEM177 deletion to assess in vivo consequences

• High-resolution imaging: Super-resolution microscopy to visualize TMEM177 localization within mitochondrial subcompartments

• Multi-omics approaches: Integration of transcriptomics, proteomics, and metabolomics data to comprehensively assess TMEM177's impact on cellular pathways

These methodological approaches would provide complementary perspectives on TMEM177's potential involvement in disease mechanisms.