TSPAN9 Antibody

What are the recommended dilution ranges for TSPAN9 antibodies across different applications?

Optimal dilutions vary by application and specific antibody. Based on compiled research data:

Methodological approach: Antibody titration is essential for each new experimental system. Begin with the manufacturer's recommended range and perform a dilution series to determine optimal signal-to-noise ratio. For Western blots, include positive controls such as platelet lysates or transfected cells expressing TSPAN9 .

What is the observed molecular weight of TSPAN9 in Western blot applications?

TSPAN9 typically appears at 27-33 kDa in reducing conditions, though the banding pattern can vary:

• The calculated molecular weight is approximately 26-27 kDa

• The observed molecular weight in human kidney tissue is approximately 27-30 kDa

• In mouse spleen tissue, TSPAN9 has been detected at 29-33 kDa

This variation reflects post-translational modifications, particularly N-linked glycosylation. TSPAN9 has a single predicted N-glycosylation site, and treatment with N-glycosidase reduces multiple bands to a single species . In overexpression systems, multiple bands representing immature glycoforms may be observed .

What tissue and cell samples have been successfully used with TSPAN9 antibodies?

TSPAN9 antibodies have been validated in various sample types:

Methodological consideration: For optimal results in new sample types, perform validation using both positive controls (tissues known to express TSPAN9, such as platelets or spleen) and negative controls (antibody pre-absorbed with immunizing peptide) .

How can I resolve multiple banding patterns when using TSPAN9 antibodies in Western blots?

Multiple bands in TSPAN9 Western blots can result from glycosylation, sample preparation issues, or antibody cross-reactivity:

• Glycosylation heterogeneity: Treat samples with N-glycosidase F to confirm glycosylation as the source of multiple bands. Research shows N-glycosidase treatment reduces multiple TSPAN9 bands to a single species .

• Sample preparation:

  • Use freshly prepared samples with protease inhibitors

  • Compare reducing vs. non-reducing conditions (tetraspanins have conserved disulfide bonds)

  • Avoid repeated freeze-thaw cycles of antibodies and samples

• Specificity verification:

  • Include peptide competition assays with the immunizing peptide as shown in validation studies with EL4 cell lysates

  • Include TSPAN9 overexpression and knockdown controls

Researchers studying TSPAN9 in platelets have successfully used non-reducing conditions with 1% Nonidet P40 and 1% dodecylmaltoside lysis buffer (to fully solubilize lipid rafts) for quantitative Western blotting .

What approaches can be used to quantitatively compare TSPAN9 expression levels across different cell types?

For quantitative TSPAN9 expression analysis, researchers have established effective protocols:

• Reference standard approach: Generate FLAG-tagged TSPAN9 reference standards by transiently transfecting HEK-293T cells, followed by lysis with combined 1% Nonidet P40 and 1% dodecylmaltoside buffer .

• Calibration curve method: Prepare serial dilutions of reference standards alongside test samples, followed by Western blotting under non-reducing conditions.

• Quantitative detection: Employ enhanced chemiluminescence (ECL) in combination with quantitative imaging systems like the GeneGnome system .

• Data normalization: Express TSPAN9 levels relative to a standard tetraspanin (e.g., CD9) or housekeeping protein.

This approach has been successfully used to determine the relative expression levels of tetraspanins (including TSPAN9) in platelets, where CD9 expression was arbitrarily set to 100 and other tetraspanins were quantified in relation to this standard .

How can I optimize co-immunoprecipitation protocols for studying TSPAN9 interactions with other membrane proteins?

Successful co-immunoprecipitation of TSPAN9 and its interaction partners requires specialized approaches for membrane proteins:

• Lysis conditions: Use mild detergents that preserve tetraspanin-enriched microdomains (TEMs):

  • For preserving stronger interactions: 1% Triton X-100

  • For preserving the entire tetraspanin web: 1% Brij 97 or CHAPS

  • For complete solubilization: Combine 1% Nonidet P40 with 1% dodecylmaltoside

• IP protocol optimization:

  • Use freshly prepared lysates supplemented with protease inhibitors

  • Perform sonication followed by centrifugation (12,000 rpm for 1 min) to clear lysates

  • For FLAG-tagged TSPAN9, use anti-FLAG M2 agarose beads with 2-hour incubation at 4°C under constant agitation

  • Wash precipitates three times with PBS before elution with sample buffer

• Validation controls:

  • Include isotype control antibodies

  • Include known TSPAN9 interaction partners as positive controls (e.g., collagen receptor GPVI, integrin α6β1)

  • Include non-interacting membrane proteins as negative controls (e.g., GPIbα, integrins αIIbβ3 or α2β1)

Researchers have successfully used this approach to demonstrate TSPAN9 interaction with ITGB1/integrin β1 in U2OS cells using FLAG-tagged TSPAN9 constructs .

What considerations should be made when using TSPAN9 antibodies for studying its role in cancer progression?

TSPAN9 appears to have context-dependent roles in cancer, requiring careful experimental design:

• Contradictory roles: Studies show TSPAN9 can have opposing functions in different cancer types:

  • Anti-oncogenic in gastric cancer

  • Pro-metastatic in osteosarcoma, promoting EMT

• Recommended approaches:

  • Use multiple antibody clones targeting different epitopes to confirm findings

  • Complement antibody-based detection with mRNA expression analysis

  • Include both gain-of-function (overexpression) and loss-of-function (shRNA knockdown) experiments

• Pathway analysis considerations:

  • Examine TSPAN9's relationship with FAK/Ras/ERK signaling, as RNA-seq analysis of TSPAN9-depleted HOS cells revealed altered expression of genes in these pathways

  • Assess EMT markers when studying TSPAN9 in cancer cells (β-Catenin, N-Cadherin, FN1, Vimentin, Snai1)

• Controls and validation:

  • For IHC studies, include both normal tissue and cancer tissue from the same organ

  • Validate antibody specificity in the specific cancer tissue being studied

What are the critical factors for successfully using TSPAN9 antibodies in immunofluorescence studies of tetraspanin microdomains?

Studying tetraspanin microdomains presents unique challenges due to their specialized membrane organization:

• Sample preparation:

  • For platelets: Fix with 2% paraformaldehyde followed by permeabilization with 0.1% Triton X-100

  • For cultured cells: Mild fixation (2-4% paraformaldehyde) is preferred to preserve membrane integrity

• Co-localization studies:

  • Include markers of tetraspanin-enriched microdomains (e.g., CD9, CD151)

  • Include TSPAN9 partners like GPVI and integrin α6β1 for platelet studies

  • Avoid harsh detergents that may disrupt microdomain organization

• Antibody selection:

  • For surface epitopes: Non-permeabilized cells with antibodies against extracellular domains

  • For intracellular epitopes: Permeabilized cells with antibodies against intracellular domains (e.g., C-terminal tail antibodies)

• Imaging considerations:

  • Super-resolution microscopy techniques (STED, STORM) provide better resolution of tetraspanin microdomains than conventional confocal microscopy

  • Use appropriate controls to establish threshold settings and account for autofluorescence

• Validation approaches:

  • Peptide competition assays with immunizing peptide

  • Comparison of staining pattern with published literature

  • Correlation with Western blot or flow cytometry results from the same samples

How should I approach the study of TSPAN9 glycosylation status and its functional implications?

TSPAN9 contains a single predicted N-linked glycosylation site that affects its molecular weight and potentially its function:

• Experimental approaches:

  • Compare native vs. deglycosylated TSPAN9 using N-glycosidase F treatment

  • Use site-directed mutagenesis to create glycosylation-deficient TSPAN9 mutants

  • Compare expression patterns of different glycoforms in various tissues and disease states

• Functional analysis:

  • Assess the impact of glycosylation on TSPAN9 protein stability and half-life

  • Evaluate whether glycosylation affects TSPAN9 trafficking to the plasma membrane

  • Determine if glycosylation modulates TSPAN9's interactions with partner proteins

• Analytical methods:

  • Use lectins in combination with TSPAN9 antibodies to characterize glycan structures

  • Employ glycoproteomics approaches to identify exact glycosylation sites and structures

  • Apply metabolic labeling with modified sugars to track newly synthesized TSPAN9 glycoforms

Research has shown that treatment of TSPAN9 immunoprecipitates with N-glycosidase reduces multiple bands to a single species, confirming the presence and impact of N-linked glycosylation .

What strategies can be employed to study TSPAN9's role in platelet function using antibody-based approaches?

TSPAN9's expression in platelets and its potential role in platelet function requires specialized methods:

• Experimental design considerations:

  • Use both resting and activated platelets (various agonists: thrombin, collagen, ADP)

  • Analyze TSPAN9 distribution in platelet fractions (membrane, cytoskeletal, soluble)

  • Examine TSPAN9 redistribution during platelet activation and spreading

• Functional approaches:

  • Employ blocking antibodies against extracellular domains to test functional effects

  • Combine with platelet aggregation and adhesion assays

  • Assess impact on platelet signaling pathways (e.g., GPVI-mediated signaling)

• Advanced techniques:

  • Flow cytometry to quantify surface vs. total TSPAN9 in platelets

  • Live-cell imaging with fluorescently-tagged anti-TSPAN9 Fab fragments

  • Super-resolution microscopy to visualize TSPAN9 distribution in tetraspanin-enriched microdomains

• Controls and validation:

  • Use platelets from other species as comparison (human vs. mouse)

  • Include other tetraspanins (CD9, CD151) as comparators

  • Correlate protein findings with TSPAN9 mRNA expression in megakaryocytes

Research has established that TSPAN9 is relatively highly expressed in the megakaryocyte/platelet lineage and co-localizes with collagen receptor GPVI (glycoprotein VI) and integrin α6β1 but not with von Willebrand receptor GPIbα or the integrins αIIbβ3 or α2β1 .

How can I use TSPAN9 antibodies to investigate its role in EMT and metastasis mechanisms?

For studying TSPAN9's involvement in epithelial-mesenchymal transition (EMT) and metastasis:

• Experimental approaches:

  • Compare TSPAN9 expression between primary tumors and metastatic lesions

  • Correlate TSPAN9 expression with EMT markers in cancer tissues

  • Assess changes in TSPAN9 expression during TGF-β-induced EMT in cell models

• Mechanistic studies:

  • Combine TSPAN9 antibodies with antibodies against signaling molecules (FAK, ERK1/2)

  • Investigate TSPAN9's relationship with integrin β1 and its activation status

  • Follow TSPAN9 redistribution during cell migration and invasion

• RNA-seq correlation:

  • Validate protein findings in the context of transcriptomic data

  • Focus on FAK/Ras/ERK pathway components identified in RNA-seq of TSPAN9-depleted cells

  • Examine correlation with EMT-related genes (β-Catenin, N-Cadherin, FN1, Vimentin, Snai1)

• Advanced analytics:

  • Use proximity ligation assays to study TSPAN9 interactions with EMT regulators

  • Apply phospho-specific antibodies to examine activation status of TSPAN9-associated signaling

  • Quantify protein levels in response to EMT inducers or inhibitors

Recent research has indicated that TSPAN9 can promote EMT and osteosarcoma metastasis, contrasting with its anti-oncogenic function in gastric cancer, highlighting the need for context-specific analysis .

What are the most effective approaches for troubleshooting weak or non-specific signals when using TSPAN9 antibodies?

When encountering signal problems with TSPAN9 antibodies:

• Weak or no signal:

  • Increase antibody concentration incrementally (follow dilution ranges in section 1.1)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Enhance signal detection methods (highly sensitive ECL reagents)

  • Confirm sample preparation preserves membrane proteins

  • Verify TSPAN9 expression in your sample type (check literature or databases)

• High background or non-specific signals:

  • Optimize blocking conditions (5% BSA often better than milk for membrane proteins)

  • Increase washing duration and number of washes

  • Decrease antibody concentration

  • Pre-absorb antibody with the immunizing peptide as a negative control

  • Test alternative detergents for membrane protein extraction

• Inconsistent results:

  • Standardize protein extraction methods

  • Prepare fresh working solutions of antibody

  • Avoid repeated freeze-thaw cycles of antibodies

  • Establish positive controls (e.g., platelets, spleen tissue)

  • Consider lot-to-lot variation in antibodies

Researchers have successfully used antibody pre-absorption with immunizing peptide as a control to demonstrate specificity, as shown in Western blot analysis of TSPAN9 in EL4 cell lysate where signal was eliminated by peptide competition .

How can I optimize TSPAN9 antibody performance for different applications?

Application-specific optimization strategies:

Human Factor considerations: When transferring protocols between species or tissue types, validate antibody performance in the new system before proceeding with experimental work.