tspan31 Antibody

What is TSPAN31 and why is it significant in cellular research?

TSPAN31 is a protein with four transmembrane domains belonging to the tetraspanin (TM4SF) family. These transmembrane proteins comprise 33 mammalian members that mediate signal transduction events involved in regulating cell development, activation, growth, and motility . TSPAN31 has gained research significance due to its role as the natural antisense transcript of cyclin-dependent kinase 4 (CDK4) and its involvement in growth-related cellular processes . Recent studies have revealed its critical regulatory functions in survival and apoptotic signals in cancer cells, making it an important target for oncology research .

What are the molecular characteristics of TSPAN31?

TSPAN31 has the following molecular characteristics:

These characteristics are essential for identifying and validating TSPAN31 in experimental settings . The discrepancy between calculated (23 kDa) and observed (30 kDa) molecular weights suggests post-translational modifications that researchers should consider when interpreting Western blot results.

What types of TSPAN31 antibodies are available for research?

Several types of TSPAN31 antibodies are available for research applications:

• Polyclonal antibodies: Such as the rabbit polyclonal IgG (21987-1-AP) that targets TSPAN31 in multiple applications including Western blot, immunohistochemistry, and ELISA .

• Monoclonal antibodies: Recombinant monoclonal antibodies like EPR11225(2) that offer higher specificity for particular epitopes of TSPAN31 .

• Recombinant protein fragments: PrEST Antigen TSPAN31, which is a recombinant protein fragment of human TSPAN31 that can be used for various applications including antibody validation .

The selection of antibody type should be based on experimental requirements, with monoclonal antibodies offering greater specificity and reproducibility for precise targeting, while polyclonal antibodies often provide higher sensitivity due to recognition of multiple epitopes.

What are the validated applications for TSPAN31 antibodies?

TSPAN31 antibodies have been validated for several applications with specific dilution recommendations:

These applications enable researchers to detect and quantify TSPAN31 in various sample types including cell lysates and tissue sections . It is recommended that researchers titrate these reagents in each testing system to obtain optimal results, as the optimal conditions can be sample-dependent.

What tissue or cell types show positive TSPAN31 expression?

TSPAN31 expression has been positively detected in:

This expression profile suggests that TSPAN31 is present in various normal tissues as well as cancer cell lines, making it relevant for both physiological and pathological studies .

How should TSPAN31 antibody samples be prepared and stored?

For optimal results, TSPAN31 antibodies should be:

• Stored at -20°C where they remain stable for one year after shipment .

• Aliquoting is unnecessary for -20°C storage for some products (such as 21987-1-AP) .

• Small volume products (20μl) may contain 0.1% BSA as a stabilizer .

• The storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

Proper storage conditions ensure antibody stability and maintain consistent experimental results over time. Researchers should avoid repeated freeze-thaw cycles which can degrade antibody quality and performance.

What signaling pathways does TSPAN31 regulate in cancer cells?

TSPAN31 has been identified as a critical regulator of multiple signaling pathways in cancer cells:

• Akt Signaling Pathway: TSPAN31 knockdown reduces the expression of phosphorylated Akt (p-Akt), phosphorylated GSK3β (p-GSK3β), and β-catenin .

• β-catenin Nuclear Translocation: TSPAN31 knockdown restrains β-catenin migration to the cell nucleus, affecting downstream transcriptional activity .

• CDK4 Regulation: As the natural antisense transcript of CDK4, TSPAN31 regulates the expression of CDK4 mRNA and protein, which impacts cell cycle progression .

• miR-135b Regulation: miR-135b can directly regulate TSPAN31 expression. When miR-135b induces TSPAN31 silencing, it increases CDK4 protein levels .

• p53 Regulation: p53 negatively regulates TSPAN31 expression, suggesting a role in tumor suppression pathways .

These regulatory mechanisms highlight TSPAN31's importance in cancer cell biology and its potential as a therapeutic target or biomarker.

How does TSPAN31 expression correlate with cancer progression?

Research demonstrates significant correlations between TSPAN31 expression and cancer progression:

• In gastric cancer:

  • TSPAN31 is highly expressed in gastric cancer tissues compared to normal tissues .

  • High expression of TSPAN31 correlates with poor prognosis in gastric cancer patients .

  • TSPAN31 regulates proliferation, migration, and apoptosis of gastric cancer cells .

• In hepatocellular carcinoma (HCC):

  • TSPAN31 knockdown significantly inhibits HCC cell invasion and migration .

  • TSPAN31 affects survival and apoptotic signals in HCC cells through modulation of the Akt signaling pathway .

• In cervical cancer:

  • TSPAN31 suppresses cell proliferation through down-regulation of its antisense pairing with CDK4 .

These findings suggest that TSPAN31 may serve as both a prognostic biomarker and a potential therapeutic target in various cancer types.

What is the relationship between TSPAN31 and other molecular factors in cancer?

TSPAN31 shows significant interactions and correlations with several molecular factors:

• CDK4: TSPAN31 is the natural antisense transcript of CDK4 and regulates its expression at both mRNA and protein levels .

• METTL1 and CCT2:

  • The expression levels of TSPAN31, METTL1, and CCT2 are positively correlated in gastric cancer cells .

  • Down-regulation of TSPAN31 can partially reverse the promoting effect of high expression of METTL1 or CCT2 on the malignant phenotype of gastric cancer cells .

• p53: Negatively regulates TSPAN31 expression, suggesting a tumor suppressive mechanism .

• miR-135b: Can directly regulate TSPAN31 expression, with miR-135b-induced TSPAN31 silencing increasing CDK4 protein levels .

These molecular relationships provide insight into the complex regulatory networks involving TSPAN31 and offer potential targets for combined therapeutic approaches.

What are the recommended antigen retrieval methods for TSPAN31 immunohistochemistry?

For optimal TSPAN31 detection in immunohistochemistry applications:

• Primary recommendation: Antigen retrieval with TE buffer at pH 9.0 .

• Alternative method: Antigen retrieval with citrate buffer at pH 6.0 .

The choice between these methods may depend on the specific tissue type and fixation conditions. Researchers should optimize antigen retrieval procedures for their particular experimental settings to achieve maximum staining sensitivity while maintaining tissue morphology.

Why might there be discrepancies between calculated and observed molecular weights of TSPAN31?

The calculated molecular weight of TSPAN31 is 23 kDa (210 amino acids), while the observed molecular weight in Western blot applications is typically around 30 kDa . This discrepancy may be attributed to:

• Post-translational modifications such as glycosylation, phosphorylation, or other covalent additions that increase the apparent molecular weight.

• The hydrophobic nature of transmembrane proteins like TSPAN31, which can affect their migration patterns in SDS-PAGE.

• Alternative splicing or the presence of isoforms, as suggested by the NCBI gene information that mentions "leaky scanning may allow translation initiation at the downstream start codon to encode an isoform (3) that has a shorter N-terminus" .

When validating TSPAN31 antibodies, researchers should consider these factors and confirm specificity through additional controls such as knockdown or knockout samples.

How can researchers validate the specificity of TSPAN31 antibodies?

To ensure the specificity of TSPAN31 antibodies, researchers should implement the following validation strategies:

• Positive controls: Using samples known to express TSPAN31, such as HeLa cells, HepG2 cells, PC-3 cells, or human/mouse skeletal muscle tissue .

• Negative controls: Including samples with TSPAN31 knockdown or knockout through siRNA or CRISPR-Cas9 technology.

• Peptide competition assays: Pre-incubating the antibody with excess TSPAN31 peptide used as the immunogen to block specific binding.

• Cross-reactivity testing: Evaluating antibody performance across multiple species if cross-reactivity is claimed.

• Multiple detection methods: Confirming TSPAN31 expression using different antibodies or detection techniques (e.g., mass spectrometry).

These validation steps are crucial for ensuring experimental reproducibility and accurate interpretation of results.

How is TSPAN31 being studied as a potential cancer biomarker or therapeutic target?

Current research is exploring TSPAN31's potential as both a biomarker and therapeutic target:

• As a prognostic biomarker:

  • Studies have correlated high TSPAN31 expression with poor prognosis in gastric cancer patients .

  • Research is investigating whether TSPAN31 expression levels could predict treatment response or disease recurrence.

• As a therapeutic target:

  • TSPAN31 knockdown studies have demonstrated inhibition of cancer cell proliferation, migration, and invasion .

  • Research is exploring whether targeting TSPAN31 could sensitize cancer cells to existing therapies.

• In combination therapies:

  • Given TSPAN31's interaction with pathways like Akt signaling and its correlation with factors like METTL1 and CCT2, studies are investigating combinatorial approaches targeting multiple pathway components simultaneously .

These research directions highlight the potential clinical applications of TSPAN31 research beyond its basic biological functions.

What are the emerging techniques for studying TSPAN31 protein interactions?

Emerging techniques for investigating TSPAN31 protein interactions include:

• Proximity-based labeling methods:

  • BioID or APEX2-based approaches to identify proteins in close proximity to TSPAN31 within living cells.

  • These techniques are particularly valuable for studying transmembrane proteins like TSPAN31 whose interactions may be disrupted by traditional lysis procedures.

• Advanced microscopy techniques:

  • Super-resolution microscopy to visualize TSPAN31 localization and co-localization with potential interaction partners.

  • FRET (Förster Resonance Energy Transfer) or BRET (Bioluminescence Resonance Energy Transfer) to detect direct protein-protein interactions.

• Crosslinking mass spectrometry:

  • Chemical crosslinking followed by mass spectrometry to capture and identify transient or weak interactions that might be missed by traditional co-immunoprecipitation.

• Organoid or 3D culture systems:

  • Studying TSPAN31 in more physiologically relevant models that better recapitulate tissue architecture and cellular interactions.

These advanced techniques may provide deeper insights into TSPAN31's function in tetraspanin-enriched microdomains (TEMs) and its role in coordinating cellular signaling events.