tmem167a Antibody

What is TMEM167A and why are antibodies against it important in research?

TMEM167A (Transmembrane Protein 167A) is a transmembrane protein associated with the Golgi apparatus and vesicular secretion processes. Research has established that TMEM167A plays critical roles in cancer biology, particularly in gliomas, by affecting the endo-lysosomal system and tumor growth . TMEM167A antibodies are essential tools for investigating this protein's expression, localization, and function in various research contexts, especially in cancer studies related to EGFR signaling pathways and p53 status.

What research applications are TMEM167A antibodies commonly used for?

TMEM167A antibodies serve multiple research applications, including:

• Western blotting (WB) for detecting and quantifying TMEM167A expression levels

• Immunohistochemistry on paraffin-embedded tissues (IHC-P) for examining tissue expression patterns

• Immunocytochemistry/Immunofluorescence (ICC/IF) for visualizing subcellular localization

• Co-immunoprecipitation for studying protein-protein interactions

Commercial antibodies like Rabbit Polyclonal TMEM167A antibodies are suitable for these applications and react with human samples . These applications allow researchers to comprehensively explore TMEM167A's role in normal physiology and disease states.

What validation methods should researchers implement for TMEM167A antibodies?

Proper validation of TMEM167A antibodies is critical for ensuring experimental reliability. Recommended validation methods include:

These validation steps are particularly important when investigating TMEM167A in different experimental models or when comparing expression levels across different tissue samples.

How can TMEM167A antibodies be used to investigate EGFR signaling pathways?

TMEM167A plays a crucial role in maintaining EGFR stability and signaling specifically in wild-type p53 tumors. Research has demonstrated that TMEM167A knockdown reduces membrane EGFR levels in p53 wild-type cells, leading to decreased EGFR signaling as evidenced by reduced AKT phosphorylation . This effect is not observed in p53 mutant cells, suggesting a p53-dependent mechanism.

For investigating this relationship, researchers can use TMEM167A antibodies to:

• Perform co-localization studies with EGFR using immunofluorescence

• Monitor changes in TMEM167A expression following EGFR activation or inhibition

• Analyze TMEM167A-EGFR interactions through proximity ligation assays

• Assess the impact of TMEM167A modulation on EGFR trafficking and degradation

These methodological approaches can help elucidate the mechanisms by which TMEM167A influences EGFR signaling and potentially identify new therapeutic targets in cancer.

What is the significance of TMEM167A in wild-type versus mutant p53 tumors?

TMEM167A expression shows a significant correlation with poor prognosis specifically in wild-type p53 gliomas but not in p53 mutant tumors. Studies have demonstrated that:

• High TMEM167A expression is associated with significantly reduced patient survival (396 days versus 837 days for low expression)

• TMEM167A expression is lower in p53 mutant tumors compared to wild-type p53 tumors

• Knockdown of TMEM167A inhibits tumor growth in p53 wild-type glioma cells but not in p53 mutant cells

To investigate this p53-dependent relationship, researchers can use TMEM167A antibodies for:

• Comparative analysis of TMEM167A expression in wild-type versus mutant p53 tumor samples

• Examining changes in TMEM167A localization and function in response to p53 activation

• Correlating TMEM167A expression with patient outcomes stratified by p53 status

• Studying the molecular mechanisms underlying the differential requirements for TMEM167A based on p53 status

This research direction may reveal important insights into tumor biology and potential therapeutic vulnerabilities.

How can researchers optimize TMEM167A antibody protocols for glioma tissue studies?

When working with glioma tissue samples, researchers should consider several technical factors to optimize TMEM167A antibody staining:

• Fixation optimization: Different fixatives may affect epitope accessibility; typically, 10% neutral buffered formalin works well for transmembrane proteins.

• Antigen retrieval methods: Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be tested to determine optimal conditions.

• Antibody concentration: Titrate antibody concentrations (typically starting at 1:100-1:500 dilutions) to identify the optimal signal-to-noise ratio.

• Incubation conditions: Extended primary antibody incubations (overnight at 4°C) often improve staining of transmembrane proteins.

• Detection systems: Use high-sensitivity detection systems for low-abundance proteins like TMEM167A.

• Controls: Include both positive controls (tissues known to express TMEM167A) and negative controls (primary antibody omission and isotype controls).

These optimizations are particularly important when working with clinical samples where TMEM167A expression may correlate with patient outcomes.

What experimental designs can elucidate TMEM167A's role in tumor growth in vivo?

TMEM167A antibodies are valuable tools for in vivo tumor growth studies. Researchers have demonstrated that TMEM167A knockdown in U87 cells (p53 wild-type) increases survival in mouse xenograft models and reduces AKT phosphorylation, confirming inhibition of EGFR signaling . Similar effects were observed in GBM4 cells that express the vIII isoform of EGFR .

For comprehensive in vivo studies, researchers should design experiments that include:

• Expression analysis: IHC staining of tumor sections to assess TMEM167A expression patterns and correlate with tumor characteristics.

• Genetic manipulation models: Comparing tumor growth between TMEM167A-expressing and TMEM167A-knockdown cells in xenograft models.

• Signaling pathway analysis: Using TMEM167A antibodies alongside phospho-specific antibodies (e.g., phospho-AKT) to assess downstream signaling in tumor samples.

• Therapeutic intervention studies: Examining how TMEM167A expression changes in response to treatments targeting EGFR or related pathways.

• Patient-derived xenografts: Testing TMEM167A expression in PDX models to better recapitulate human disease.

Such experimental designs can provide mechanistic insights into TMEM167A's role in tumor biology and potentially identify new therapeutic strategies.

How should researchers interpret subcellular localization patterns of TMEM167A?

TMEM167A has been associated with the Golgi apparatus and vesicular secretion processes . When interpreting immunofluorescence staining patterns:

Changes in localization patterns in response to experimental manipulations may indicate dynamic regulation of TMEM167A function. For instance, EGFR activation might alter TMEM167A distribution, providing insights into its role in receptor trafficking.

What technical considerations are important for Western blot analysis of TMEM167A?

Western blot analysis of TMEM167A requires careful optimization due to its nature as a transmembrane protein. Key considerations include:

• Protein extraction: Use lysis buffers containing appropriate detergents (e.g., RIPA buffer with protease and phosphatase inhibitors) for efficient membrane protein solubilization .

• Sample preparation: Avoid excessive heating of samples, which can cause aggregation of membrane proteins.

• Gel percentage: Use 8-10% acrylamide gels for optimal resolution of TMEM167A.

• Transfer conditions: Optimize transfer time and voltage for efficient transfer of membrane proteins to nitrocellulose.

• Blocking agents: Test different blocking agents (BSA vs. non-fat milk) to minimize background while preserving specific signals.

• Controls: Include positive controls (cells known to express TMEM167A) and loading controls (β-actin, GAPDH) for accurate comparison .

• Antibody dilution: Determine optimal primary antibody concentration through titration experiments.

These technical considerations ensure reliable and reproducible detection of TMEM167A in Western blot analyses.

How can TMEM167A antibodies be used to study the relationship between TMEM167A and autophagy?

Research suggests that TMEM167A may be involved in selective autophagosome formation during ferroptosis rather than starvation-induced autophagy . To investigate this relationship, researchers can employ TMEM167A antibodies in conjunction with autophagy markers through:

• Co-localization studies: Examine the spatial relationship between TMEM167A and autophagy markers (LC3, ATG5) during ferroptosis induction.

• Flux analysis: Assess how TMEM167A knockdown affects autophagy flux by monitoring LC3-II turnover in the presence of lysosomal inhibitors.

• Immunoprecipitation: Identify potential interactions between TMEM167A and autophagy machinery components.

• Live-cell imaging: Track dynamic changes in TMEM167A localization during autophagy induction using fluorescently tagged antibodies.

• Immunoelectron microscopy: Precisely localize TMEM167A in relation to autophagosomal structures.

These methodological approaches can help elucidate how TMEM167A contributes to autophagy regulation, particularly in the context of ferroptosis.

What methods can resolve contradictory findings about TMEM167A function across different cancer types?

Research on TMEM167A has primarily focused on gliomas, but functions may vary across cancer types. To address potential contradictions, researchers should:

• Use multiple antibody validation approaches: Confirm antibody specificity across different cell types and tissue samples.

• Employ genetic models: Compare results from antibody-based studies with genetic knockdown/knockout approaches.

• Context-specific analysis: Evaluate TMEM167A function in relation to p53 status, EGFR expression, and other relevant factors across cancer types.

• Domain-specific antibodies: Utilize antibodies targeting different epitopes to identify potential isoforms or post-translational modifications.

• Multi-omics integration: Correlate antibody-based findings with transcriptomic, proteomic, and phosphoproteomic data to obtain a comprehensive view of TMEM167A function.

This systematic approach can help reconcile seemingly contradictory findings and establish a more complete understanding of TMEM167A's context-dependent functions.

How can researchers investigate potential TMEM167A interaction partners using antibody-based approaches?

To identify and characterize TMEM167A interaction partners, researchers can implement several antibody-based strategies:

• Co-immunoprecipitation (Co-IP): Use TMEM167A antibodies to pull down the protein complex and identify interacting partners through mass spectrometry.

• Proximity ligation assay (PLA): Detect in situ protein-protein interactions between TMEM167A and candidate partners with single-molecule sensitivity.

• FRET/FLIM analysis: Examine direct protein interactions using fluorescently labeled antibodies against TMEM167A and potential partners.

• Cross-linking coupled to IP: Stabilize transient interactions before immunoprecipitation to capture weak or dynamic interactors.

• Two-hybrid screening validation: Confirm interactions identified through screening methods using antibody-based approaches.

These techniques can uncover novel TMEM167A interactions, particularly with components of the EGFR signaling pathway or proteins involved in vesicular trafficking, providing insights into its cellular functions.