TMEM140 Antibody

What is the basic structure and localization of TMEM140 protein?

TMEM140 (transmembrane protein 140) in humans is a membrane-localized protein with 185 amino acid residues and a molecular mass of approximately 20.4 kDa. The protein undergoes post-translational modifications, most notably glycosylation, which can affect antibody recognition in various applications. TMEM140 gene orthologs have been identified across multiple species including mouse, rat, bovine, frog, chimpanzee, and chicken, which provides options for comparative studies using species-specific antibodies .

What is the genomic context of TMEM140 and its relevance to disease research?

The TMEM140 gene is located on chromosome 7q33, a region frequently associated with various cancer types. This genomic positioning is particularly significant as alterations in chromosome 7 are closely linked to the development of glioma and other malignancies. Understanding this context is essential when designing research studies targeting TMEM140 expression in pathological conditions, particularly in neuro-oncology research .

How does TMEM140 expression vary across normal and pathological tissues?

TMEM140 exhibits significantly elevated expression in glioma tissues compared to normal brain tissue. Expression levels positively correlate with glioma histological grade, tumor size, and inversely correlate with patient survival rates. These expression patterns make TMEM140 a potential biomarker for disease progression and prognosis in glioma research. When designing immunohistochemistry experiments, researchers should account for this differential expression when selecting appropriate positive and negative control tissues .

What criteria should researchers consider when selecting a TMEM140 antibody?

When selecting a TMEM140 antibody, researchers should evaluate:

• Application compatibility (ELISA, IHC, IF, WB)

• Species reactivity (human-specific vs. cross-reactive)

• Clonality (monoclonal for specificity vs. polyclonal for broader epitope recognition)

• Conjugation requirements (unconjugated vs. fluorophore/enzyme conjugates)

• Validated epitope regions (considering potential splice variants)

• Batch-to-batch consistency documentation

• Publication record in similar experimental contexts

The choice between conjugated (FITC, HRP, biotin) and unconjugated antibodies should be dictated by the specific experimental design rather than convenience .

What validation steps are essential before using TMEM140 antibodies in critical experiments?

A comprehensive validation protocol should include:

• Western blot analysis to confirm specificity at the expected molecular weight (20.4 kDa)

• Positive control testing in tissues with known high TMEM140 expression (e.g., glioma tissue)

• Negative control testing in tissues with minimal expression

• Peptide competition assays to confirm epitope specificity

• siRNA knockdown controls to verify antibody specificity

• Comparison of staining patterns across multiple antibodies targeting different TMEM140 epitopes

• Cross-validation across multiple detection methods (IHC, IF, WB)

This systematic approach prevents misleading results from antibody cross-reactivity, particularly important when studying membrane proteins with structural similarities .

How can TMEM140 antibodies be effectively employed in studying the protein's role in cancer cell biology?

TMEM140 antibodies can be strategically deployed in several advanced experimental paradigms:

• Co-immunoprecipitation studies: To identify interaction partners involved in adhesion and apoptotic pathways.

• Chromatin immunoprecipitation: For researchers investigating transcriptional regulation of TMEM140.

• Proximity ligation assays: To visualize and quantify protein-protein interactions in situ.

• Live-cell imaging with fluorescently-conjugated antibodies: For trafficking and localization studies.

• Tissue microarray analysis: For high-throughput screening across multiple patient samples.

These applications can reveal molecular mechanisms through which TMEM140 regulates cell adhesion molecules (ICAM1, VCAM1, syndecan1) and apoptotic proteins (caspase-3, Bax, Bcl2) in cancer models .

What considerations are important when using TMEM140 antibodies for signaling pathway investigations?

When investigating TMEM140's involvement in signaling pathways:

• Use phospho-specific antibodies alongside TMEM140 antibodies to track activation states

• Employ temporal analysis after stimulus application (e.g., growth factors, stress inducers)

• Consider subcellular fractionation before immunoblotting to distinguish membrane-bound vs. cytosolic pools

• Implement multiplexed analysis with markers for adhesion and apoptotic pathways

• Validate findings with pharmacological inhibitors of suspected pathway components

Research indicates TMEM140 influences multiple signaling molecules involved in cell survival and adhesion, making pathway analysis complex but informative .

What are the optimal protocols for immunohistochemical detection of TMEM140 in tissue samples?

The optimal IHC protocol for TMEM140 detection includes:

• Tissue preparation: Formalin-fixed paraffin-embedded (FFPE) sections (4-6 μm) or frozen sections

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

• Blocking: 5-10% normal serum in PBS with 0.1% Triton X-100 for 1 hour

• Primary antibody incubation: Anti-TMEM140 (1:100-1:500 dilution) overnight at 4°C

• Detection system: Polymer-based detection systems show superior signal-to-noise ratio

• Counterstaining: Light hematoxylin counterstaining to avoid obscuring membrane staining

• Controls: Include glioma tissue as positive control and normal brain tissue as negative/low expression control

This protocol optimizes detection while minimizing background, crucial for accurate quantification of TMEM140 expression levels in correlation studies with clinical parameters .

What experimental design is recommended for studying TMEM140 function using knockdown/knockout approaches?

A comprehensive experimental design should include:

• Multiple silencing approaches:

  • siRNA (transient): Using at least two independent sequences targeting different regions

  • shRNA (stable): For long-term studies requiring sustained knockdown

  • CRISPR-Cas9: For complete knockout studies

• Essential controls:

  • Non-targeting siRNA/shRNA controls

  • Rescue experiments with exogenous TMEM140 expression

  • Isogenic cell line pairs (wildtype vs. knockout)

• Functional readouts:

  • Adhesion assays (measuring attachment to extracellular matrix components)

  • Migration and invasion assays (Boyden chamber/transwell assays)

  • Apoptosis measurements (Annexin V/PI staining, caspase activity assays)

  • Cell cycle analysis (PI staining, EdU incorporation)

Previous research demonstrates that TMEM140 knockdown significantly affects cell adhesion molecules and apoptotic regulators, providing a framework for experimental validation .

How should researchers address variability in TMEM140 antibody staining patterns across different sample types?

To address staining variability:

• Technical normalization approaches:

  • Implement batch correction using technical replicates

  • Utilize automated staining platforms to reduce technical variability

  • Incorporate digital image analysis with standardized algorithms

• Biological considerations:

  • Account for tissue heterogeneity through microdissection or single-cell approaches

  • Consider the influence of post-translational modifications on epitope availability

  • Evaluate the impact of tissue fixation duration on membrane protein antigenicity

• Statistical handling:

  • Apply appropriate statistical tests for non-parametric distributions

  • Implement mixed-effects models to account for intra- and inter-sample variability

  • Use bootstrapping approaches for robust confidence interval estimation

These strategies help distinguish biological significance from technical artifacts when analyzing TMEM140 expression across experimental conditions .

How can researchers reconcile conflicting data between TMEM140 protein levels and functional outcomes?

When facing discrepancies between protein expression and functional outcomes:

• Consider post-translational regulation: Examine glycosylation state and other modifications

• Evaluate protein localization: Membrane-bound vs. internalized TMEM140 may have different functions

• Assess temporal dynamics: Expression timing may be as important as absolute levels

• Investigate compensatory mechanisms: Related transmembrane proteins may provide functional redundancy

• Examine context-dependency: Microenvironmental factors may modulate TMEM140 function

• Analyze isoform-specific effects: Different splice variants may have opposing functions

A comprehensive approach incorporating these considerations allows for nuanced interpretation of seemingly contradictory experimental outcomes in TMEM140 research .

What are the established functional roles of TMEM140 in glioma pathobiology?

Current research has established several critical roles for TMEM140 in glioma:

• Cell adhesion regulation: TMEM140 modulates expression of adhesion molecules ICAM1, VCAM1, and syndecan1, affecting tumor cell invasion and migration

• Apoptosis modulation: TMEM140 knockout upregulates pro-apoptotic proteins (caspase-3, Bax) and downregulates anti-apoptotic proteins (Bcl2)

• Correlation with clinical parameters: TMEM140 expression positively correlates with tumor size, histological grade, and inversely with patient survival

• Therapeutic target potential: Inhibition of TMEM140 reduces adhesion and metastasis while promoting apoptosis in glioma models

These findings establish TMEM140 as a multifunctional protein central to glioma progression and a potential therapeutic target .

What emerging methodological approaches might advance TMEM140 research?

Emerging approaches with potential to advance TMEM140 research include:

• Single-cell proteomics: To examine TMEM140 expression heterogeneity within tumors

• Spatially-resolved transcriptomics: To correlate TMEM140 with microenvironmental features

• Cryo-electron microscopy: For structural analysis of TMEM140 and its interaction partners

• CRISPR activation/interference screens: To identify synthetic lethal interactions in TMEM140-high tumors

• Patient-derived organoids: For functional validation in more physiologically relevant models

• Targeted protein degradation approaches: As potential therapeutic strategies beyond knockdown

• AI-assisted image analysis: For automated quantification of TMEM140 expression patterns

These methodologies could overcome current technical limitations and provide deeper mechanistic insights into TMEM140 biology .