TSPAN11 Antibody

What is TSPAN11 and why is it a target of research interest?

TSPAN11 (Tetraspanin-11) is a membrane protein belonging to the tetraspanin superfamily, characterized by four conserved transmembrane domains with intracellular N- and C-termini and two extracellular domains . Tetraspanins are involved in diverse cellular processes including cell activation, proliferation, adhesion, motility, differentiation, and cancer development . Recent research suggests TSPAN11 may play significant roles in development through regulation of integrin function and in autoimmune diseases by affecting immune cell functions and interactions with proteins that regulate immune responses and inflammation . These biological functions make TSPAN11 an important target for researchers studying cell signaling, development, and immune system regulation.

What is the molecular weight of TSPAN11 and why does it sometimes differ from predictions?

The calculated molecular weight of TSPAN11 is approximately 28 kDa, but the experimentally observed molecular weight in Western blot analyses typically ranges from 35-38 kDa . This discrepancy between predicted and observed molecular weights is common for membrane proteins and is often attributed to post-translational modifications such as glycosylation. When running Western blots for TSPAN11, researchers should be prepared for bands appearing at the 35-38 kDa range rather than at the calculated 28 kDa. This information is crucial for correctly identifying TSPAN11 in your experimental samples and avoiding misinterpretation of results.

How do I select the appropriate TSPAN11 antibody for my research?

When selecting a TSPAN11 antibody, consider:

• Species reactivity: Determine if the antibody reacts with your species of interest. Available TSPAN11 antibodies have been validated for human and mouse samples .

• Application compatibility: Different antibodies are optimized for specific applications:

  • For Western blot: Select antibodies validated for WB with recommended dilutions typically ranging from 1:500-1:2000 .

  • For Immunofluorescence: Choose antibodies validated for ICC/IF with recommended dilutions between 0.25-2 μg/mL .

  • For Immunohistochemistry: Use antibodies specifically validated for IHC .

• Clonality: Most available TSPAN11 antibodies are rabbit polyclonal antibodies , which offer good sensitivity but might have batch-to-batch variability.

• Immunogen: Consider the immunogen used to generate the antibody. Some TSPAN11 antibodies target specific peptide sequences (e.g., "RLSDELKQHLNRTLAENYGQPGATQITASVDRLQQDFKCCGSN" ), while others target recombinant protein fragments (e.g., amino acids 100-200 of the human protein ).

• Validation data: Review available validation data including Western blot images, immunofluorescence patterns, and reactivity with positive control samples.

What are the optimal conditions for Western blot analysis using TSPAN11 antibodies?

For optimal Western blot results with TSPAN11 antibodies:

• Sample preparation:

  • Use tissues with known TSPAN11 expression such as brain tissue (human or mouse) or cell lines like RT4 (human urinary bladder cancer), U-251 MG (human brain glioma), or SH-SY5Y (human neuroblastoma) .

  • Ensure complete lysis with membrane-compatible detergents to extract this transmembrane protein effectively.

• Antibody dilutions:

  • Most TSPAN11 antibodies work well at dilutions between 1:500-1:1000 for Western blot .

  • For specific antibodies like ab221740, a dilution of 1:250 has been successfully used .

• Detection of bands:

  • Expect bands at 35-38 kDa, rather than the calculated 28 kDa molecular weight .

  • Multiple bands may indicate different isoforms or post-translational modifications.

• Controls:

  • Include positive controls such as human brain tissue lysate or mouse brain tissue lysate .

  • Consider using recombinant TSPAN11 protein or cells transfected with TSPAN11 expression constructs as additional controls .

How can I optimize immunofluorescence staining with TSPAN11 antibodies?

For successful immunofluorescence (ICC/IF) experiments:

• Fixation method:

  • PFA-fixation followed by Triton X-100 permeabilization has been successfully used with TSPAN11 antibodies .

  • Alternative fixation methods might be necessary depending on your specific cell type.

• Antibody concentration:

  • Use TSPAN11 antibodies at 0.25-2 μg/mL for immunofluorescence .

  • Some specific antibodies like ab221740 have been used at 4 μg/ml in ICC/IF applications .

• Cell types:

  • SH-SY5Y cells (human neuroblastoma) have shown detectable TSPAN11 expression and successful staining .

  • For other cell types, optimization might be necessary.

• Expected pattern:

  • As a membrane protein, TSPAN11 should predominantly show membrane localization, possibly with some cytoplasmic signal.

  • Verify specificity with appropriate controls, including secondary antibody-only controls.

• Co-localization studies:

  • Consider co-staining with markers for specific cellular compartments to better characterize TSPAN11 localization.

What controls should I include when working with TSPAN11 antibodies?

Including appropriate controls is essential for validating your TSPAN11 antibody results:

• Positive tissue/cell controls:

  • Human or mouse brain tissue for WB and IHC applications .

  • Cell lines with known TSPAN11 expression: SH-SY5Y, RT4, or U-251 MG cells .

• Negative controls:

  • Secondary antibody-only controls to assess non-specific binding.

  • Tissues or cells known to have low or no TSPAN11 expression.

• Blocking peptide controls:

  • When available, use the immunizing peptide to pre-absorb the antibody as a specificity control.

• Genetic controls:

  • Cells with TSPAN11 knockdown (siRNA or CRISPR) as negative controls.

  • Cells overexpressing TSPAN11 (using available cDNA clones ) as positive controls.

• Cross-validation:

  • When possible, validate findings using multiple antibodies targeting different epitopes of TSPAN11.

  • Confirm protein-level findings with mRNA expression data.

Why might I see unexpected bands or no signal in my TSPAN11 Western blot?

Several factors can cause issues with TSPAN11 Western blots:

For membrane proteins like TSPAN11, sample preparation is particularly critical. Ensure you're using appropriate detergents to solubilize membrane proteins and consider deglycosylation experiments if glycosylation is affecting band patterns.

How can I enhance the specificity of immunostaining with TSPAN11 antibodies?

To improve specificity in immunofluorescence or immunohistochemistry:

• Optimize antibody dilution: Start with the recommended range (0.25-2 μg/mL ) and perform titration experiments to find the optimal concentration that maximizes specific signal while minimizing background.

• Blocking optimization:

  • Test different blocking solutions (BSA, serum, commercial blockers).

  • Extend blocking time if background is high.

• Fixation method comparison:

  • Compare different fixatives (PFA, methanol, acetone).

  • Adjust permeabilization conditions for membrane proteins.

• Antigen retrieval:

  • For IHC, test different antigen retrieval methods if signal is weak.

• Signal amplification:

  • Consider using signal amplification systems for low-abundance targets.

• Validation with controls:

  • Always run parallel staining with TSPAN11-positive and negative samples.

  • Include peptide competition assays where applicable.

Can TSPAN11 antibodies be recycled, and what are the limitations?

While antibody recycling is sometimes practiced to reduce costs, it's generally not recommended for TSPAN11 antibodies for several reasons:

• Efficiency concerns: Recycled antibodies typically show reduced performance with each use .

• Buffer system alterations: After initial use, the buffer composition changes, potentially affecting antibody stability and performance .

• Storage variability: Inconsistent storage conditions between uses can further reduce antibody effectiveness .

• Limited recycling potential: Laboratory testing suggests that only high-titer antibodies can be recycled, and even then, typically only up to three times .

• Protocol for attempted recycling: If recycling is necessary due to limited resources:

  • After incubation, recover unused antibody by centrifugation.

  • Store at 4°C.

  • Use within one week.

  • Expect reduced performance with each subsequent use .

Given these limitations, for critical experiments and reproducible results, fresh antibody aliquots are strongly recommended over recycled antibodies.

How can TSPAN11 antibodies be used to investigate protein-protein interactions?

TSPAN11 antibodies can be valuable tools for studying protein-protein interactions through several approaches:

• Co-immunoprecipitation (Co-IP):

  • Use TSPAN11 antibodies to pull down TSPAN11 and associated proteins.

  • This approach is particularly relevant since TSPAN11 may interact with proteins involved in immune responses and inflammation .

  • When performing Co-IP with membrane proteins like TSPAN11, carefully select lysis conditions that maintain protein-protein interactions while solubilizing membrane complexes.

• Proximity ligation assay (PLA):

  • Combine TSPAN11 antibodies with antibodies against suspected interaction partners.

  • PLA can detect proteins in close proximity (<40 nm), suggesting potential interactions.

  • This technique is particularly useful for studying membrane protein interactions in their native cellular context.

• Immunofluorescence co-localization:

  • Use TSPAN11 antibodies in combination with antibodies against potential interacting proteins.

  • Advanced microscopy techniques like FRET or FLIM can provide additional evidence of protein proximity.

• Membrane complex isolation:

  • Tetraspanins often form specialized membrane microdomains termed "tetraspanin-enriched microdomains" (TEMs).

  • TSPAN11 antibodies can help isolate and characterize these complexes to identify novel interaction partners.

• Functional validation:

  • Combine antibody-based interaction studies with functional assays to understand the biological significance of identified interactions, particularly in contexts related to integrin function and immune response regulation .

What approaches can be used to investigate TSPAN11's role in immune regulation?

Given TSPAN11's potential role in autoimmune diseases and immune regulation , several research approaches using TSPAN11 antibodies can be employed:

• Expression profiling in immune cells:

  • Use TSPAN11 antibodies for Western blot, flow cytometry, or immunofluorescence to characterize expression patterns across immune cell populations.

  • Compare expression levels between resting and activated immune cells.

  • Analyze expression in samples from patients with autoimmune conditions versus healthy controls.

• Functional blocking studies:

  • If available, use function-blocking TSPAN11 antibodies to inhibit its activity in immune cell assays.

  • Measure effects on cell proliferation, cytokine production, and cell-cell interactions.

• Signaling pathway analysis:

  • Use TSPAN11 antibodies in combination with phospho-specific antibodies to investigate how TSPAN11 affects immune signaling pathways.

  • Perform immunoprecipitation followed by mass spectrometry to identify TSPAN11-associated signaling complexes in immune cells.

• In vivo models:

  • Use TSPAN11 antibodies for immunohistochemistry in tissues from autoimmune disease models.

  • Investigate co-localization with inflammatory markers and immune cell infiltrates.

• Correlation with clinical parameters:

  • In translational research, correlate TSPAN11 expression (detected by antibodies) with disease severity, treatment response, or specific clinical features in autoimmune conditions.

How can I integrate TSPAN11 antibody-based assays with genomic and transcriptomic approaches?

An integrated multi-omics approach can provide comprehensive insights into TSPAN11 biology:

• Validation of genomic findings:

  • Use TSPAN11 antibodies to verify protein-level consequences of genetic variants identified in genomic studies.

  • Investigate whether SNPs associated with diseases affect TSPAN11 protein expression, localization, or function.

• Correlation between mRNA and protein expression:

  • Compare TSPAN11 mRNA levels (from RNA-seq or qPCR) with protein levels (detected by antibodies) across tissues or experimental conditions.

  • Investigate potential post-transcriptional regulatory mechanisms if discrepancies are observed.

• ChIP-seq integration:

  • Combine TSPAN11 antibody-based protein studies with ChIP-seq data of transcription factors regulating TSPAN11 expression.

  • This approach can help elucidate the transcriptional control mechanisms of TSPAN11.

• Single-cell analysis:

  • Correlate single-cell RNA-seq data with single-cell protein data using TSPAN11 antibodies for techniques like CITE-seq or imaging mass cytometry.

  • This can reveal cell-specific expression patterns and potential functional heterogeneity.

• Multi-omics data integration:

  • Create comprehensive models of TSPAN11 regulation and function by integrating antibody-based protein data with genomic, transcriptomic, and epigenomic datasets.

  • Network analysis can reveal TSPAN11's position in broader biological pathways, particularly in development and immune regulation contexts .

What are the optimal storage conditions for TSPAN11 antibodies?

Proper storage of TSPAN11 antibodies is crucial for maintaining their activity and specificity:

Following these storage guidelines will help ensure consistent performance of TSPAN11 antibodies across experiments and extend their useful lifespan.