TSPAN18 Antibody

What is TSPAN18 and what are its key structural features?

TSPAN18 (tetraspanin 18) is a membrane protein belonging to the tetraspanin (TM4SF) superfamily, characterized by four conserved transmembrane regions. In humans, the canonical protein consists of 248 amino acid residues with a molecular mass of approximately 27.7 kDa . TSPAN18 undergoes post-translational modifications, particularly glycosylation, which may affect its functionality and detection in experimental systems. The protein's subcellular localization is primarily in the plasma membrane, consistent with its role in membrane-associated signaling complexes .

What cellular functions has TSPAN18 been implicated in?

TSPAN18 has been identified as a novel regulator of thrombo-inflammation, which is the interplay between thrombosis and inflammation that causes acute organ damage in conditions such as ischemic stroke and venous thrombosis . Research has demonstrated that TSPAN18 partners with store-operated Ca²⁺ signaling components and uniquely activates the Ca²⁺-responsive NFAT transcription factor in an Orai1-dependent manner . Additionally, TSPAN18 has been implicated in cellular processes including cell activation, proliferation, adhesion, motility, and differentiation, which are common functions across the tetraspanin family .

What is the expression pattern of TSPAN18 across different tissues?

According to RT-PCR and RNA-Seq data analysis, TSPAN18 demonstrates a relatively specific expression pattern. It is predominantly expressed in human endothelial cells but not in most other cell types . Analysis of mouse single-cell RNA-Seq data across 20 organs reveals that 9 of the 14 most highly TSPAN18-expressing cell types are endothelial cells . Beyond endothelial expression, TSPAN18 has been detected in human platelets through proteomics approaches and in human and mouse platelets and megakaryocytes at the mRNA level . The TSPAN18 marker can also be used to identify Medulla Oblongata Splatter Neurons .

What criteria should researchers consider when selecting a TSPAN18 antibody?

When selecting a TSPAN18 antibody, researchers should consider several critical factors:

• Application compatibility: Verify the antibody has been validated for your specific application (WB, ELISA, IF, etc.)

• Species reactivity: Ensure reactivity with your experimental model (human, mouse, rat)

• Epitope location: Consider whether targeting specific domains (e.g., center region) is important for your research

• Conjugation requirements: Determine if unconjugated or conjugated (FITC, HRP, Biotin, APC) formats are needed

• Validation evidence: Review available data showing specificity and performance in relevant contexts

The search results indicate numerous commercial antibodies with different specifications. For example, some antibodies target the middle or center region of TSPAN18, while others have varying reactivity profiles across species .

How can researchers validate the specificity of TSPAN18 antibodies?

Validating TSPAN18 antibody specificity is crucial for reliable experimental results. A comprehensive validation approach should include:

• Positive control selection: Use tissues known to express TSPAN18, such as human placenta tissue which has been validated for Western blot applications

• Molecular weight verification: Confirm detection at the expected molecular weight (approximately 28-29 kDa as observed in validated samples)

• Knockout/knockdown controls: Compare staining between wild-type and TSPAN18-knockout or knockdown samples

• Cross-reactivity assessment: Test the antibody against related tetraspanin family members to ensure specificity

• Multiple detection methods: Validate using orthogonal techniques (e.g., mass spectrometry) to confirm target identity

For TSPAN18, validation can be challenging due to its relatively specific expression pattern, making appropriate positive control selection particularly important.

What are the optimal conditions for Western blot detection of TSPAN18?

For successful Western blot detection of TSPAN18, researchers should follow these methodological recommendations:

• Sample preparation: Use tissues with known TSPAN18 expression (e.g., human placenta) or endothelial cell lysates

• Antibody dilution: Employ a dilution range of 1:1000-1:4000 as recommended for validated antibodies

• Expected band size: Look for a band at approximately 28 kDa, which corresponds to the observed molecular weight of TSPAN18

• Buffer conditions: Use PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 for antibody storage

• Storage temperature: Maintain antibody at -20°C for optimal stability (stable for one year after shipment)

The detection of TSPAN18 may be sample-dependent, so researchers should check validation data galleries when available and optimize conditions for their specific experimental system .

How can TSPAN18 be effectively detected in endothelial cells?

Given TSPAN18's predominant expression in endothelial cells, the following methodological approach is recommended:

• Cell line selection: Choose appropriate endothelial cell models that express TSPAN18 based on RT-PCR or RNA-Seq data

• Application selection: Consider immunofluorescence (IF) for localization studies or Western blot for protein level assessment

• Controls: Include positive controls (endothelial cells) and negative controls (cell types with minimal TSPAN18 expression)

• Co-staining: Combine with endothelial markers (e.g., CD31) to confirm cell-type specificity

• Signaling pathway analysis: Consider co-staining with Orai1 or other Ca²⁺ signaling components to investigate functional interactions

Since TSPAN18 partners with Orai1 in endothelial cells, co-immunoprecipitation experiments can be valuable for investigating this interaction in various endothelial contexts .

How can TSPAN18 antibodies be used to investigate its role in thrombo-inflammation?

To investigate TSPAN18's role in thrombo-inflammation, researchers can employ these advanced approaches:

• Endothelial activation models: Use TSPAN18 antibodies to assess protein levels during inflammatory activation of endothelial cells

• Calcium signaling studies: Combine TSPAN18 immunodetection with calcium imaging to correlate expression with functional outcomes

• Co-localization analysis: Perform high-resolution imaging to examine TSPAN18 co-localization with Orai1 and other components of store-operated calcium entry

• In vivo models: Use TSPAN18 antibodies for immunohistochemistry in mouse models of thrombo-inflammatory conditions

• Comparative analysis: Compare TSPAN18 expression and localization between normal and pathological samples

Research has demonstrated that TSPAN18 activates the Ca²⁺-responsive NFAT transcription factor by 20-fold in an Orai1-dependent manner, suggesting it plays a significant role in calcium-dependent inflammatory processes .

What experimental approaches can address the relationship between TSPAN18 and platelet function?

While Tspan18-knockout mice do not show major platelet dysfunction, subtle roles in platelet Ca²⁺ signaling merit investigation through these approaches:

• Calcium flux measurement: Compare Ca²⁺ responses between wild-type and Tspan18-deficient platelets using fluorescent indicators

• Aggregation assays: Employ in vitro aggregation assays with varying agonist concentrations to detect subtle differences

• Signaling pathway analysis: Use phospho-specific antibodies to examine downstream effectors of calcium signaling

• Receptor clustering studies: Investigate whether TSPAN18 affects the organization of platelet receptors using super-resolution microscopy

• In vivo thrombosis models: Assess the contribution of platelet TSPAN18 using chimeric mice with selective platelet deficiency

Research has shown that Tspan18-knockout platelets exhibit defective aggregation in response to collagen-related peptides, suggesting a subtle but potentially important role in platelet function under specific conditions .

How is TSPAN18 implicated in neuropsychiatric disorders?

TSPAN18 gene mutations have been associated with schizophrenia in Han Chinese populations . Researchers investigating this connection should consider:

• Genetic analysis: Sequence TSPAN18 in patient cohorts to identify potentially pathogenic variants

• Expression studies: Compare TSPAN18 levels in brain tissue or derived cells from patients versus controls

• Functional assays: Assess how disease-associated mutations affect TSPAN18's interaction with Orai1 and calcium signaling

• Animal models: Generate knock-in mice carrying human disease-associated variants for behavioral and molecular phenotyping

• Drug response correlation: Investigate whether TSPAN18 variants correlate with treatment responses in schizophrenia

The association with schizophrenia suggests TSPAN18 may play roles in neurodevelopment or neuronal function that extend beyond its well-characterized endothelial functions.

What is the potential of TSPAN18 as a therapeutic target?

TSPAN18 represents a promising therapeutic target for several reasons:

• Tissue specificity: Its relatively endothelial-specific expression pattern may allow targeted intervention without systemic effects

• Pathway selectivity: Targeting TSPAN18 could enable Orai1 inhibition in an endothelial-specific manner, avoiding toxicity that would result from inhibiting this important Ca²⁺ channel in all cells

• Disease relevance: Its role in thrombo-inflammation makes it relevant for conditions like ischemic stroke and venous thrombosis

• Precedent: Targeting tetraspanins has precedent, as antibodies to tetraspanin CD37 are in clinical trials for chronic lymphocytic leukemia

• Expanding indications: TSPAN18 may be relevant for endothelial-driven inflammatory diseases, cancer angiogenesis, and retinal neovascularization

Research strategies should focus on developing highly specific inhibitors or modulators of TSPAN18 function that preserve the essential roles of Orai1 in other tissues.

How can researchers overcome difficulties in detecting low-abundance TSPAN18?

When investigating tissues or cell types with low TSPAN18 expression, consider these methodological approaches:

• Sample enrichment: Isolate specific cell populations (e.g., endothelial cells) from heterogeneous tissues

• Signal amplification: Utilize tyramide signal amplification or other enhancement techniques for immunohistochemistry

• Sensitive detection methods: Employ highly sensitive techniques like droplet digital PCR for mRNA detection

• Optimized lysis conditions: Test different lysis buffers to ensure complete extraction of membrane-embedded tetraspanins

• Antibody combinations: Use cocktails of antibodies targeting different epitopes to improve detection sensitivity

The table below presents recommended approaches based on expression levels:

What approaches can resolve discrepancies in TSPAN18 detection between different antibodies?

When facing inconsistent results between different TSPAN18 antibodies, implement this systematic troubleshooting approach:

• Epitope mapping: Determine which protein regions are targeted by each antibody

• Isoform awareness: Check if discrepancies might result from detection of different TSPAN18 isoforms

• Post-translational modifications: Assess whether glycosylation or other modifications might mask epitopes in certain contexts

• Denaturation sensitivity: Test both denaturing and non-denaturing conditions as some epitopes may be conformation-dependent

• Validation hierarchy: Establish a hierarchy of validation methods to resolve conflicts (e.g., knockout controls > peptide competition > independent antibodies)

Researchers should maintain awareness that the 267 amino acid calculated molecular weight differs slightly from the observed 28 kDa molecular weight in Western blots, which may reflect post-translational modifications .

How might single-cell approaches advance our understanding of TSPAN18 biology?

Single-cell technologies offer powerful opportunities for TSPAN18 research:

• Heterogeneity mapping: Single-cell RNA-Seq can identify specific endothelial subpopulations with highest TSPAN18 expression

• Co-expression networks: Correlate TSPAN18 expression with other genes to identify potential functional networks

• Spatial transcriptomics: Map TSPAN18 expression within tissue microenvironments to understand contextual regulation

• Temporal dynamics: Analyze expression changes during development or disease progression at single-cell resolution

• Functional genomics: Combine with CRISPR screens to identify genetic modifiers of TSPAN18 function

Analysis of existing single-cell RNA-Seq data from 20 mouse organs has already revealed that 9 of the 14 most highly TSPAN18-expressing cell types are endothelial cells, highlighting the power of this approach .

What novel techniques might enhance TSPAN18 protein interaction studies?

Advanced techniques for studying TSPAN18 protein interactions include:

• Proximity labeling: BioID or APEX2 fusions with TSPAN18 to identify proximal proteins in living cells

• FRET/BRET sensors: Develop fluorescence or bioluminescence resonance energy transfer systems to monitor TSPAN18-Orai1 interactions in real-time

• Super-resolution microscopy: Nanoscale imaging of TSPAN18 distribution and co-localization with partners

• Crosslinking mass spectrometry: Identify direct interaction surfaces between TSPAN18 and its binding partners

• Cryo-electron microscopy: Structural determination of TSPAN18 in complex with Orai1 or other partners

These approaches could help elucidate how TSPAN18 uniquely regulates Orai1 function compared to other tetraspanins, potentially through conformational changes that have been hypothesized based on current research .