tspan9 Antibody

What is TSPAN9 and what cellular functions has it been implicated in?

TSPAN9, also known as NET5 or Tetraspan NET-5, is a membrane protein of the tetraspanin superfamily characterized by four conserved transmembrane regions. Tetraspanins broadly participate in diverse cellular processes including cell activation, proliferation, adhesion, motility, differentiation, and cancer progression .

TSPAN9 shows relatively specific expression in the megakaryocyte/platelet lineage compared to other blood cell types and appears to play a regulatory role in platelet function in concert with other platelet tetraspanins and their associated proteins . Recent research has also identified TSPAN9 as a potential oncogenic factor in osteosarcoma, where it promotes cell proliferation, epithelial-mesenchymal transition (EMT), and metastasis through interaction with integrin β1 and subsequent activation of the FAK-Ras-ERK1/2 signaling pathway .

What types of TSPAN9 antibodies are available for research applications?

Several TSPAN9 antibodies are commercially available with different specifications:

Researchers have also successfully generated antibodies by raising them in rabbit and chicken against the intracellular C-terminal tail of human TSPAN9 . This approach worked specifically for TSPAN9 but may not be universally applicable to all tetraspanins, as some have extremely short cytoplasmic domains that are insufficient as immunogens .

What are the validated applications for TSPAN9 antibodies?

TSPAN9 antibodies have been validated for multiple research applications:

• Western Blot (WB): Recommended dilutions range from 1:200-1:1000 . TSPAN9 is typically observed at 29-33 kDa despite a calculated molecular weight of 27 kDa .

• Immunohistochemistry (IHC-P): Successfully applied at 1:200 dilution for formalin-fixed, paraffin-embedded tissues, with confirmed reactivity in human lung carcinoma and mouse spleen tissue .

• Flow Cytometry: Validated at 1:100 dilution with secondary antibody incubation for 40 minutes, as demonstrated in human U937 cells .

• Immunoprecipitation (IP): Effective using 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate, with validated results in mouse spleen tissue .

• ELISA: Applications have been reported though specific protocols may vary .

What positive controls are recommended for TSPAN9 antibody validation?

Based on validated tissue and cell sources, the following positive controls are recommended:

• Tissues: Mouse spleen tissue, rat spleen tissue, and human lung carcinoma tissue have all demonstrated positive TSPAN9 expression .

• Cells: Human U937 cells and mouse peripheral blood leukocytes show detectable TSPAN9 expression .

• Cell lines: Human osteosarcoma cell line HOS exhibits high TSPAN9 expression compared to normal osteoblast cell lines (hFOB1.19) .

What are the recommended protocols for immunohistochemical detection of TSPAN9?

For successful immunohistochemical detection of TSPAN9 in formalin-fixed, paraffin-embedded tissues:

• Use a 1:200 dilution of TSPAN9 antibody (e.g., ab215432) .

• Apply standard antigen retrieval techniques appropriate for formalin-fixed tissues.

• Incubate with the primary antibody according to the manufacturer's recommended conditions.

• Follow with conjugation to an appropriate secondary antibody.

• Develop using DAB (3,3'-diaminobenzidine) staining for visualization .

Validated tissues include human lung carcinoma and mouse spleen, which have demonstrated specific staining patterns .

What is the optimal protocol for detecting TSPAN9 via flow cytometry?

For flow cytometric analysis of TSPAN9:

• Prepare single-cell suspensions following standard protocols for the cell type of interest.

• Incubate cells with TSPAN9 antibody at 1:100 dilution for approximately 40 minutes .

• Wash cells thoroughly with an appropriate buffer.

• Incubate with a fluorophore-conjugated secondary antibody (e.g., Goat Anti-Rabbit IgG PE conjugated) for 40 minutes .

• Wash cells and analyze by flow cytometry, comparing to appropriate control cells.

This approach has been successfully applied to human U937 cells, demonstrating specific staining compared to control populations .

How should researchers approach Western blot analysis of TSPAN9?

For optimal Western blot detection of TSPAN9:

• Prepare protein lysates using buffers suitable for membrane proteins, potentially including mild detergents to facilitate extraction.

• Load adequate protein (typically 20-50 μg per lane) and separate by SDS-PAGE.

• Transfer proteins to an appropriate membrane (PVDF or nitrocellulose).

• Block according to standard protocols.

• Incubate with TSPAN9 antibody at dilutions between 1:200-1:1000 .

• Apply appropriate secondary antibody and develop using standard chemiluminescence or fluorescence detection methods.

Researchers should note that while the calculated molecular weight of TSPAN9 is 27 kDa, the observed molecular weight typically ranges from 29-33 kDa, possibly due to post-translational modifications .

How can TSPAN9 antibodies be used to study tetraspanin microdomains?

TSPAN9 has been identified as a component of tetraspanin microdomains on the platelet surface . Researchers can use TSPAN9 antibodies to study these microdomains through:

• Co-immunoprecipitation: TSPAN9 antibodies can be used to immunoprecipitate TSPAN9 and its associated proteins from surface-biotinylated cells, which helps identify other components of tetraspanin microdomains .

• Biochemical analysis: Specific detergent conditions can be used to preserve or disrupt tetraspanin microdomains, allowing researchers to analyze the composition and stability of these structures.

• Immunofluorescence microscopy: TSPAN9 antibodies have been successfully used for immunofluorescence microscopy to visualize TSPAN9 expression and localization in megakaryocytes and platelets .

These approaches can provide valuable insights into the role of TSPAN9 in organizing and regulating the function of tetraspanin microdomains.

What approaches can be used to study TSPAN9's role in cancer progression?

Recent research has implicated TSPAN9 in cancer progression, particularly in osteosarcoma (OS). Researchers can investigate TSPAN9's oncogenic potential using:

• Expression analysis: TSPAN9 is significantly upregulated in OS tissues compared to normal osteoblast cells (fold-change = 2.131, P = 0.0037) and in OS cell lines compared to mesenchymal stem cells (fold-change = 1.9787, P = 0.0037) . Antibodies can be used to assess protein expression levels across different cancer types and stages.

• Functional studies using knockdown/overexpression:

  • siRNA-mediated knockdown of TSPAN9 in HOS cells significantly decreased cell survival and colony formation .

  • Stable shRNA knockdown of TSPAN9 can be used to assess effects on cell migration and invasion in wound-healing and Transwell assays .

  • Overexpression studies can complement knockdown approaches to confirm phenotypic effects.

• Molecular pathway analysis: TSPAN9 promotes OS progression through interaction with integrin β1, leading to activation of the FAK-Ras-ERK1/2 signaling pathway . Antibodies can be used in co-immunoprecipitation and signaling studies to further elucidate these mechanisms.

How can researchers validate TSPAN9 antibody specificity?

Ensuring antibody specificity is crucial for reliable research outcomes. Methods to validate TSPAN9 antibody specificity include:

• RNA interference: Knockdown TSPAN9 expression using siRNA constructs (e.g., siTspan9#1, siTspan9#2, siTspan9#3) and confirm reduced protein detection by Western blot or immunocytochemistry using the antibody .

• Stable knockdown validation: Generate cells stably expressing shTspan9 constructs and confirm reduced antibody reactivity compared to control (shNC) cells .

• Overexpression controls: Create cells overexpressing TSPAN9 (confirmed by GFP expression if using a GFP-tagged construct) and verify increased antibody reactivity .

• Tissue distribution analysis: Compare antibody reactivity across tissues with known TSPAN9 expression patterns, such as platelets and megakaryocytes (high expression) versus other blood cell types (lower expression) .

What are potential challenges in detecting TSPAN9 and how can they be addressed?

Detecting membrane proteins like TSPAN9 can present specific challenges:

• Membrane protein extraction: Tetraspanins are embedded in the membrane and associated with larger proteins in tetraspanin microdomains, which can make extraction and detection difficult .

  • Solution: Use mild detergents tailored for membrane protein extraction, adjusting lysis conditions to balance protein extraction with preservation of native conformation.

• Epitope accessibility: The small size of tetraspanins and their association with larger proteins may render epitopes inaccessible .

  • Solution: For antibody production, target accessible regions like the C-terminal tail rather than transmembrane domains . For detection, optimize antigen retrieval methods.

• Evolutionary conservation: Tetraspanin protein sequences are highly conserved during evolution, resulting in few non-self epitopes for antibody generation .

  • Solution: Consider using antibodies raised against synthetic peptides or fusion proteins rather than whole cells as immunogens.

• Molecular weight variation: The observed molecular weight (29-33 kDa) differs from the calculated value (27 kDa) .

  • Solution: Be aware of this discrepancy when analyzing Western blot results and consider potential post-translational modifications.

How can researchers differentiate between TSPAN9 and other tetraspanin family members?

Distinguishing TSPAN9 from other tetraspanins requires careful consideration:

• Antibody selection: Choose antibodies validated for specificity against TSPAN9, ideally with demonstrated lack of cross-reactivity with other tetraspanins.

• Expression pattern analysis: TSPAN9 shows relatively specific expression in the megakaryocyte/platelet lineage compared to other blood cell types , which can serve as a distinguishing feature.

• Molecular techniques: Complement antibody-based detection with molecular techniques like RT-PCR using primers specific to TSPAN9.

• Knockdown validation: Perform siRNA knockdown of TSPAN9 and confirm that antibody signal is reduced, supporting specificity of detection.

How can TSPAN9 antibodies be used to identify potential biomarkers in disease?

TSPAN9 shows promise as a biomarker in certain conditions:

• Cancer diagnosis and prognosis: ROC curve analyses from the GSE33383 and GSE42352 datasets indicate that TSPAN9 can serve as a valuable biomarker capable of differentiating between osteosarcoma tumors or cells and corresponding normal control samples .

• Expression correlation: Researchers can use TSPAN9 antibodies to examine protein expression across patient samples and correlate with clinical parameters, disease progression, or treatment response.

• Multimarker panels: TSPAN9 detection could be incorporated into broader panels of markers for improved diagnostic or prognostic accuracy.

What are emerging methods for studying TSPAN9 protein interactions?

Advanced techniques to study TSPAN9 interactions include:

• Proximity ligation assays: These can visualize and quantify interactions between TSPAN9 and potential binding partners like integrin β1 with higher sensitivity than conventional co-localization microscopy.

• Mass spectrometry-based interactomics: Immunoprecipitation of TSPAN9 followed by mass spectrometry analysis can identify novel interaction partners in different cellular contexts.

• Live-cell imaging: Combining TSPAN9 antibodies with advanced microscopy techniques allows visualization of dynamic protein interactions in living cells.

• CRISPR-based approaches: Gene editing of TSPAN9 combined with antibody-based detection of interacting proteins can provide insights into the functional consequences of these interactions.