TMEM176A Antibody

What is TMEM176A and why are antibodies against it important in research?

TMEM176A is a transmembrane protein that has been implicated in several pathological processes, particularly in cancer and immune regulation. The protein consists of 235 amino acids with a molecular weight of approximately 26-28 kDa, and contains multiple transmembrane domains .

TMEM176A antibodies are critical research tools because:

• They enable detection and quantification of TMEM176A expression in various tissues and cell types

• They help elucidate TMEM176A's role in normal physiology and disease states

• They facilitate the study of TMEM176A's involvement in key cellular pathways, particularly ERK signaling

• They allow investigation of TMEM176A's function in dendritic cell maturation and immune regulation

Research has shown that TMEM176A expression is frequently dysregulated in certain cancers, making it a potential biomarker and therapeutic target, particularly in lung cancer and hepatocellular carcinoma .

What are the main applications of TMEM176A antibodies in experimental systems?

TMEM176A antibodies can be utilized across multiple experimental platforms:

For optimal results, researchers should validate antibody performance in their specific experimental systems before conducting comprehensive studies. Antigen retrieval methods significantly impact detection sensitivity in IHC applications, with Tris-EDTA buffer (pH 9.0) generally providing better results than citrate buffer (pH 6.0) .

How should researchers choose between monoclonal and polyclonal TMEM176A antibodies?

The selection between monoclonal and polyclonal antibodies depends on experimental goals:

Monoclonal TMEM176A Antibodies:

• Provide high specificity and reproducibility

• Example: Mouse monoclonal antibody against full-length recombinant TMEM176A (AA 1-235)

• Ideal for applications requiring consistent lot-to-lot performance

• Better for detecting specific epitopes in their native conformation

• Typically used when comparing TMEM176A expression levels across multiple samples

Polyclonal TMEM176A Antibodies:

• Recognize multiple epitopes on the TMEM176A protein

• Examples: Rabbit polyclonal antibodies targeting different regions

• Offer higher sensitivity, particularly useful in low-expression contexts

• More tolerant of protein denaturation, making them versatile for various applications

• Preferable for initial characterization studies or when protein structure may be altered

For methylation studies in cancer tissues, polyclonal antibodies may provide better detection across heterogeneously methylated samples, while monoclonal antibodies might offer more consistent results for quantitative analyses .

What are appropriate positive and negative controls for TMEM176A antibody validation?

Proper controls are essential for accurately interpreting TMEM176A antibody results:

Recommended Positive Controls:

• Fetal human brain tissue for Western blot

• Mouse brain tissue for immunohistochemistry

• HeLa cells for immunofluorescence

• Lung cancer cell lines with known TMEM176A expression (e.g., H1299, H23)

Recommended Negative Controls:

• Primary antibody omission

• Isotype control antibodies (IgG1 for mouse monoclonal antibodies)

• Cell lines with TMEM176A knockdown

• Tissue samples with confirmed TMEM176A methylation (for expression studies)

For comprehensive validation, researchers should include both technical controls (antibody specificity) and biological controls (tissues/cells with known expression patterns). When studying methylation effects, unmethylated and methylated lung cancer samples can serve as biological controls .

What are common technical challenges when working with TMEM176A antibodies?

Researchers frequently encounter several challenges when working with TMEM176A antibodies:

• Non-specific binding: Particularly problematic in tissues with high background. Solution: Optimize blocking conditions and antibody dilutions; consider using highly validated antibodies like Prestige Antibodies .

• Variable epitope accessibility: TMEM176A is a multi-pass membrane protein, making some epitopes difficult to access. Solution: Test antibodies targeting different regions (N-terminal, internal, C-terminal) .

• Detection in fixed tissues: Formalin fixation can mask epitopes. Solution: Implement appropriate antigen retrieval methods, particularly heat-mediated retrieval with Tris-EDTA buffer (pH 9.0) .

• Methylation interference: TMEM176A promoter methylation can affect expression levels, complicating antibody-based detection. Solution: Consider complementing protein detection with methylation analysis .

• Cross-reactivity with TMEM176B: TMEM176A and TMEM176B share sequence homology. Solution: Validate antibody specificity using recombinant protein controls .

How can TMEM176A antibodies contribute to cancer research?

TMEM176A antibodies serve as valuable tools in cancer research, particularly for:

• Diagnostic biomarker development: TMEM176A methylation is found in 53.66% of primary lung cancers, making it a potential diagnostic marker . Antibodies can help correlate methylation status with protein expression.

• Therapeutic target validation: Research has shown that restoration of TMEM176A expression induces cancer cell apoptosis and G2/M phase arrest while inhibiting colony formation, proliferation, migration, and invasion . Antibodies can monitor TMEM176A expression following experimental interventions.

• Pathway analysis: TMEM176A suppresses tumor growth by inhibiting ERK signaling. Antibodies can be used in combination with phospho-ERK antibodies to study this regulatory relationship .

• Drug sensitivity studies: Methylation of TMEM176A sensitizes cancer cells to AZD0156, an ATM inhibitor. Antibodies can help identify tumors likely to respond to such targeted therapies .

• Xenograft model evaluation: TMEM176A has been shown to suppress H1299 cell xenograft growth in mice. Antibodies can monitor TMEM176A expression in these models .

For cancer research applications, combining antibody-based detection with methylation analysis provides the most comprehensive understanding of TMEM176A's role in tumorigenesis and potential therapeutic applications.

What methodologies combine TMEM176A antibodies with methylation studies?

Integrating antibody detection with methylation analysis requires a multi-faceted approach:

• Sequential analysis workflow:

  • Assess methylation status using methylation-specific PCR (MSP)

  • Determine protein expression using validated TMEM176A antibodies

  • Correlate methylation patterns with protein expression levels

• Experimental design considerations:

  • Include cell lines with known methylation status as controls

  • Use demethylating agents (e.g., 5-aza-2'-deoxycytidine) to restore expression

  • Confirm specificity with multiple antibodies targeting different epitopes

• Recommended protocols:

  • For immunohistochemistry: Use heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0)

  • For methylation analysis: Perform bisulfite sequencing or MSP

  • For functional studies: Combine with ERK signaling pathway analysis

This integrated approach has successfully demonstrated that TMEM176A expression is regulated by promoter region methylation in lung cancer, with significant implications for understanding cancer biology and developing targeted therapies .

How do TMEM176A antibodies facilitate research on immune regulation and dendritic cell function?

TMEM176A and its related protein TMEM176B are increasingly recognized as important regulators of dendritic cell maturation and immune responses:

• Dendritic cell maturation studies:

  • TMEM176A and TMEM176B have been shown to prevent dendritic cell maturation

  • Antibodies can track expression levels during maturation processes

  • Immunofluorescence using TMEM176A antibodies can visualize subcellular localization changes during activation

• Immune dysfunction in spinal cord injury (SCI):

  • TMEM176A and TMEM176B are overexpressed in subjects with spinal cord injury

  • These proteins may inhibit protective immune responses in SCI

  • Antibodies can be used to monitor expression in peripheral blood mononuclear cells

• Therapeutic development approaches:

  • Targeting TMEM176A could promote immune system-mediated neuroprotection

  • Antibodies can assess the efficacy of interventions aimed at modulating TMEM176A expression

  • Combined with functional assays, antibody-based detection helps establish mechanism of action

Researchers studying neuroinflammation or neuroimmune interactions should consider incorporating TMEM176A antibodies into their experimental design, particularly when investigating dendritic cell function in the context of central nervous system injuries .

What emerging applications of TMEM176A antibodies should researchers consider?

Several promising research directions for TMEM176A antibodies are emerging:

• Synthetic lethality therapeutics: TMEM176A methylation status sensitizes cancer cells to ATM inhibitors like AZD0156. Antibodies could be developed as companion diagnostics to identify patients likely to respond to such targeted therapies .

• Neuroprotection strategies: Given TMEM176A's role in immune regulation after spinal cord injury, antibodies could facilitate the development of immune-based therapies to promote tissue regeneration and limit further damage in chronic SCI .

• Single-cell protein profiling: Advances in single-cell technologies could incorporate TMEM176A antibodies to understand cellular heterogeneity in cancer and immune responses.

• Spatial transcriptomics integration: Combining TMEM176A antibody-based protein detection with spatial transcriptomics could reveal location-dependent regulation in complex tissues.

• Post-translational modification studies: Developing antibodies specific to modified forms of TMEM176A could reveal regulatory mechanisms beyond methylation-controlled expression.

How can researchers effectively validate new hypotheses about TMEM176A function?

To rigorously test novel hypotheses about TMEM176A function, researchers should implement comprehensive validation strategies:

• Multiple antibody approach:

  • Use antibodies targeting different TMEM176A epitopes

  • Compare results from different antibody classes (monoclonal vs. polyclonal)

  • Validate with both commercial and custom-developed antibodies

• Complementary technique integration:

  • Combine protein detection (antibody-based) with gene expression analysis

  • Correlate with methylation studies in relevant contexts

  • Support with functional assays (e.g., proliferation, apoptosis, migration)

• Model system diversity:

  • Test hypotheses across multiple cell lines

  • Validate in primary patient samples

  • Confirm in appropriate animal models

• Pathway analysis validation:

  • Assess effects on known interacting pathways (particularly ERK signaling)

  • Perform co-immunoprecipitation to identify binding partners

  • Utilize inducible expression systems to study temporal dynamics