TSPAN33 Antibody

What is TSPAN33 and what are the most effective antibodies for its detection?

TSPAN33 is a 32 kDa protein member of the tetraspanin superfamily (TM4SF) with four transmembrane domains and a characteristic cysteine-rich long extracellular loop (LEL). It plays roles in B cell activation, macrophage function, and kidney physiology .

For effective detection, consider:

While several commercially available antibodies work effectively, validation in your specific experimental system is crucial, as TSPAN33 expression is highly tissue and activation-state specific .

What tissues and cell types express TSPAN33, and how should antibody applications be optimized?

TSPAN33 shows a restricted expression pattern with specific physiological and pathological contexts:

For optimal antibody application:

• For flow cytometry: Co-staining with CD19 and appropriate activation markers is recommended for B cell studies

• For IHC: Epitope retrieval is essential; use TE buffer pH 9.0 or citrate buffer pH 6.0

• For Western blot: Be aware of the significantly higher observed molecular weight (64-72 kDa) compared to calculated (32 kDa)

How does TSPAN33 expression change upon B cell activation and what antibody approaches best capture this dynamic?

TSPAN33 serves as a specific marker for B cell activation, with expression dramatically increasing upon stimulation:

To capture this dynamic:

• Use flow cytometry with PE-conjugated TSPAN33 antibodies for quantitative assessment

• Establish appropriate time points (12-120 hours post-stimulation)

• Include controls for B cell activation (CD40, CD69, CD86)

• For mechanistic studies, consider dual staining with phospho-specific antibodies targeting relevant B cell signaling pathways

How can TSPAN33 antibodies be utilized to study B cell migration and membrane dynamics?

TSPAN33 regulates B-lymphocyte cytoskeleton and plasma membrane-related phenomena, including protrusion formation, adhesion, phagocytosis, and cell motility . Advanced applications include:

Membrane Dynamics Analysis:

• Immunofluorescence co-localization with membrane microdomain markers

• Live-cell imaging with non-perturbing fluorescently-labeled TSPAN33 antibody fragments

• Atomic force microscopy combined with TSPAN33 immunolabeling to correlate membrane tension with protein distribution

Migration Studies Methodology:

• Establish stable TSPAN33-overexpressing or knockdown B cell lines

• Perform chemotaxis assays with CXCL12/13

• Conduct invasion assays through extracellular matrix

• Quantify fibronectin-induced spreading and measure membrane roughness and tension

• Use TSPAN33 antibodies to validate expression levels and localization

Key findings show that TSPAN33 overexpression inhibits changes in roughness and membrane tension during fibronectin-induced spreading, while enhancing migration and invasion capabilities. TSPAN33 knockdown produces opposite phenotypes .

What are the technical considerations for using TSPAN33 antibodies in lymphoma diagnosis and classification?

TSPAN33 shows differential expression across lymphoma subtypes, making it valuable for diagnostic applications:

Technical Considerations:

• Tissue Processing: Formalin-fixed paraffin-embedded (FFPE) tissues require specific epitope retrieval methods with TE buffer pH 9.0

• Antibody Selection: Use validated antibodies with confirmed specificity in lymphoma tissues

• Controls: Include normal lymphoid tissue with activated germinal centers as positive control

• Multiplex Approach: Combine with CD20, CD30, and other lymphoma markers for improved classification

• Quantitative Analysis: Consider digital pathology approaches for objective assessment of staining intensity and distribution

TSPAN33 antibody staining could complement conventional diagnostic markers, especially for differentiating activated B-cell derived lymphomas.

How do different fixation and permeabilization protocols affect TSPAN33 antibody accessibility and epitope integrity?

TSPAN33's complex membrane topology with four transmembrane domains and two extracellular loops presents unique challenges for immunodetection:

Fixation Considerations:

• Paraformaldehyde (2-4%): Preserves structure but may mask transmembrane epitopes

• Methanol/acetone: Improves accessibility to intracellular domains but can disrupt conformational epitopes in the extracellular loops

• Light fixation (0.5-1% PFA): Recommended for flow cytometry when detecting surface epitopes

Permeabilization Optimization:

• For extracellular loop epitopes: Mild detergents (0.1% saponin) or no permeabilization

• For intracellular epitopes: Stronger detergents (0.1-0.5% Triton X-100)

• For multi-epitope detection: Sequential staining approach, first targeting extracellular domains before permeabilization

Advanced Protocol for Comprehensive Epitope Analysis:

• Perform non-permeabilized surface staining with antibodies against extracellular domains

• Fix with 2% PFA for 10 minutes

• Permeabilize with 0.1% saponin

• Stain with antibodies against intracellular domains

• Validate with domain-specific antibodies targeting different regions of TSPAN33

This approach allows for studying both surface expression and intracellular pools of TSPAN33, which may have distinct functions in regulating B cell activation and membrane dynamics.

What are the methodological approaches for studying TSPAN33 interactions with other membrane proteins using antibody-based techniques?

TSPAN33 interactions with other membrane proteins, particularly integrins and ADAM10, are critical for understanding its function in membrane organization:

Co-immunoprecipitation Strategies:

• Use mild lysis conditions (1% Brij97 or CHAPS) to preserve tetraspanin-enriched microdomains

• Perform sequential immunoprecipitation to distinguish direct from indirect interactions

• Include appropriate controls (isotype, tetraspanin-free regions)

• Western blot for candidate interacting proteins (integrins, ADAM10)

Proximity Ligation Assay (PLA) Protocol:

• Fix cells with 4% PFA for 15 minutes

• Block with 5% BSA in PBS for 1 hour

• Co-incubate with anti-TSPAN33 antibody (1:100) and antibody against potential interacting partner

• Perform PLA according to manufacturer's protocol

• Quantify interaction signals in different subcellular compartments and activation states

Crosslinking Mass Spectrometry Approach:

• Treat intact cells with membrane-permeable crosslinkers

• Lyse cells and immunoprecipitate TSPAN33 complexes

• Perform on-bead digestion and analyze by LC-MS/MS

• Validate novel interactions with traditional biochemical approaches

This comprehensive approach helps decipher the TSPAN33 interactome and its role in regulating B cell membrane dynamics, particularly in the context of altered expression in lymphomas and autoimmune conditions .