TSG101 Monoclonal Antibody

What is TSG101 and why is it an important research target?

TSG101 (Tumor susceptibility gene 101 protein) is essential for endosomal sorting, membrane receptor degradation, and the final stages of cytokinesis. It plays a crucial role in cell proliferation and cell survival . TSG101 has been identified as a candidate tumor suppressor gene and belongs to the ubiquitin-conjugating enzyme family . Its importance as a research target stems from its involvement in:

• The ESCRT-I complex, which regulates vesicular trafficking processes

• Binding to ubiquitinated cargo proteins for sorting into multivesicular bodies (MVBs)

• Viral budding processes, particularly for retroviruses

• Exosome biogenesis and release

• Cell growth regulation and differentiation

• Tumor suppression mechanisms

What is the molecular weight of TSG101 and how is it detected in experimental systems?

TSG101 has a calculated molecular weight of 44 kDa and is typically observed at 43-46 kDa in Western blot analyses . The protein is sometimes detected as a doublet, which is consistent with what has been described in the literature . This may be due to internal initiation at Met 10 or post-translational modifications .

In experimental systems, TSG101 can be detected by:

• Western blotting (WB): Most commonly used, detecting bands at 44-46 kDa

• Immunohistochemistry (IHC): For tissue sections

• Immunofluorescence (IF): For cellular localization studies

• Flow cytometry: For quantitative analysis of cellular expression

• Immunoprecipitation (IP): For protein-protein interaction studies

What tissues and cell lines express TSG101?

TSG101 is widely expressed across multiple tissues and cell types:

What are the recommended dilutions and protocols for using TSG101 antibodies in different applications?

Based on validated protocols, the following dilutions are recommended for TSG101 antibodies:

Protocol for Western Blot using TSG101 antibody:

• Separate proteins on a 5-20% SDS-PAGE gel at 70V (stacking gel)/90V (resolving gel) for 2-3 hours

• Transfer proteins to a nitrocellulose membrane at 150 mA for 50-90 minutes

• Block the membrane with 5% non-fat milk/TBS for 1.5 hour at room temperature

• Incubate with TSG101 antibody at appropriate dilution overnight at 4°C

• Wash with TBS-0.1% Tween 3 times for 5 minutes each

• Probe with appropriate HRP-conjugated secondary antibody

• Develop signal using enhanced chemiluminescence detection

How can I optimize antigen retrieval for TSG101 immunohistochemistry?

Optimizing antigen retrieval is critical for successful TSG101 immunohistochemistry:

• Buffer selection: TE buffer pH 9.0 is suggested as the primary choice for antigen retrieval with TSG101 antibodies. Alternatively, citrate buffer pH 6.0 may be used if TE buffer results are suboptimal .

• Heat-mediated retrieval: Heat-mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) has been validated for rabbit monoclonal antibodies against TSG101 .

• Temperature and duration: For optimal results, perform heat-mediated antigen retrieval at 100°C for 20-30 minutes followed by cooling to room temperature.

• Optimization strategy: It is recommended to test both pH conditions (pH 6.0 and pH 9.0) and varying retrieval times (10, 20, and 30 minutes) to determine optimal conditions for your specific tissue sample and antibody .

How can TSG101 antibodies be used to confirm exosome isolation?

TSG101 is a well-established exosomal marker used to confirm successful exosome isolation:

• Antibody selection: Use rabbit anti-TSG101 antibodies that recognize the human form of the protein for exosome detection .

• Multi-marker approach: For comprehensive exosome characterization, use TSG101 antibodies in conjunction with antibodies against other exosome markers such as:

  • Tetraspanins (CD9, CD63, and CD81)

  • Heat shock protein HSP70

• Western blot protocol for exosome validation:

  • Lyse isolated exosomes in RIPA buffer with protease inhibitors

  • Separate proteins by SDS-PAGE

  • Transfer to PVDF or nitrocellulose membrane

  • Block with 5% non-fat milk

  • Probe with anti-TSG101 antibody (1:1000-1:5000 dilution)

  • Detect with appropriate secondary antibody and visualization system

  • Expected band at approximately 44-46 kDa confirms presence of TSG101-positive exosomes

How does TSG101 interact with viral proteins and what implications does this have for virology research?

TSG101 plays a critical role in viral budding through specific protein-protein interactions:

• HIV-1 interaction mechanism: TSG101 interacts with the p6 region of HIV-1 Gag protein, specifically through the PTAPP motif. This interaction is essential for viral particle budding .

• Experimental validation: The interaction between TSG101 and Gag has been demonstrated through:

  • Yeast two-hybrid assays

  • Immune capture assays

  • Co-immunoprecipitation experiments

• Competitive inhibition: Addition of a peptide containing the PTAPP motif (ALQSRPEPTAPPEES) causes reduction in TSG101 capture by Pr55 Gag, while a mutant LIAPP sequence shows no effect, confirming specificity .

• Research applications: Understanding this interaction is valuable for:

  • Developing antiviral strategies targeting TSG101-viral protein interactions

  • Studying virus assembly and budding mechanisms

  • Identifying cellular factors that influence viral replication

What is the relationship between TSG101 genetic variants and HIV disease progression?

Research has identified important relationships between TSG101 genetic variants and HIV disease progression:

• Haplotype influence: Two polymorphic sites in the TSG101 5′ area (positions −183 and +181 relative to translation start) specify three haplotypes (A, B, and C) occurring at frequencies of 67%, 21%, and 12% respectively. Haplotype C is associated with relatively rapid AIDS progression, while haplotype B is associated with slower disease progression .

• CD4 T-cell decline: TSG101 variants show differential effects on CD4 T-cell decline rates:

  • A/C-C/C (susceptible group): Fastest CD4 decline

  • A/A-B/C (neutral group): Intermediate CD4 decline

  • A/B-B/B (protective group): Slowest CD4 decline

• Viral load dynamics: Significant differences in viral load increase over time have been observed between the protective and susceptible TSG101 genotypic groups (P < 0.0001) .

• Mechanism hypothesis: These genetic variations may affect the functional activity of TSG101 protein in viral budding, thereby altering levels of circulating virus in the blood and influencing disease progression .

How does TSG101 influence the MDM2-p53 pathway in cancer research?

TSG101 has been shown to have a regulatory relationship with MDM2 and p53:

• TSG101-MDM2 interaction: The Ubc domain of TSG101 interferes with ubiquitination of MDM2, inhibiting MDM2 decay and elevating its steady-state level .

• Effect on MDM2 degradation: TSG101 inhibits MDM2 degradation and prolongs its half-life. The half-life of MDM2 nearly doubled (from approximately 15 min to 28 min) in cells overexpressing TSG101 .

• Impact on p53 levels: Overproduction of TSG101 in cotransfected cells is associated with further elevation of the MDM2 protein level and a prominent further decrease in p53 .

• Research implications: This TSG101/MDM2 regulatory loop provides insights into:

  • Cancer cell proliferation mechanisms

  • Tumor suppression pathways

  • Potential therapeutic targets in the ubiquitin-proteasome system

What strategies can improve specificity and reduce background in TSG101 immunostaining?

To improve specificity and reduce background in TSG101 immunostaining:

• Antibody titration: Perform careful titration experiments to determine optimal antibody concentration. For immunofluorescence applications, test dilutions from 1:200 to 1:4000 to find the optimal signal-to-noise ratio .

• Blocking optimization: Use 5% non-fat milk or 0.1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for optimal blocking before antibody incubation .

• Washing protocols: Implement thorough washing steps with PBS-Tween (0.1% Tween-20 in PBS) between antibody incubations.

• Fixation methods: Compare 4% paraformaldehyde fixation versus 100% methanol fixation (5 min) to determine which yields better results for your specific application .

• Permeabilization conditions: Test different permeabilization reagents and times:

  • 0.1% Triton X-100 for 5 minutes

  • 0.1% saponin with 10% normal goat serum and 0.75% glycine

• Negative controls: Always include appropriate negative controls (secondary antibody alone, isotype controls) to assess non-specific binding .

Why might I observe a doublet pattern for TSG101 in Western blotting and how should this be interpreted?

The doublet pattern frequently observed for TSG101 in Western blotting has specific biological origins:

• Known phenomenon: TSG101 is consistently detected as a doublet in Western blot analyses, which is well-documented in the literature .

• Biological explanation: This doublet pattern may arise from:

  • Internal initiation at Met 10 resulting in a slightly truncated protein

  • Post-translational modifications such as phosphorylation

  • Alternative splicing variants (TSG101 has 2 isoforms produced by alternative splicing with molecular masses of 44 and 32 kDa)

• Interpretation guidelines:

  • The primary band should appear at 43-46 kDa

  • The second band commonly appears slightly lower

  • Both bands represent TSG101 protein and should be considered in quantification

  • Consistent doublet pattern across samples indicates proper antibody specificity

• Verification approach: If doublet authenticity is in question, validation can be performed using:

  • siRNA knockdown of TSG101 (both bands should diminish)

  • Comparison with alternative TSG101 antibodies recognizing different epitopes

How can TSG101 antibodies be used to study exosome-mediated intercellular communication?

TSG101 antibodies serve as critical tools for studying exosome-mediated communication:

• Exosome characterization: TSG101 antibodies enable confirmation of exosome identity and purity in isolation protocols, which is essential for downstream functional studies .

• Research applications:

  • Multi-omics approaches: TSG101 antibodies can be used to validate exosomes in studies examining how pancreatic cancer cell extracellular vesicles mediate the unfolded protein response in normal pancreatic epithelial cells .

  • Therapeutic RNA delivery: TSG101 antibodies help characterize self-assembled small RNAs as a new generation of RNAi therapeutics delivered via extracellular vesicles .

  • Brain-targeted delivery: TSG101 antibody-validated extracellular vesicles have been studied for delivery of anti-miR-106b to inhibit morphine-induced primary ciliogenesis in the brain .

  • Senescence communication: Validation of extracellular vesicles from senescent stem cells that trigger adaptive mechanisms in young stem cells by increasing antioxidant enzyme expression .

• Methodological approach: For comprehensive exosome studies:

  • Isolate exosomes using ultracentrifugation, precipitation, or size-exclusion chromatography

  • Validate using Western blot with TSG101 and other exosome markers (CD9, CD63, CD81, HSP70)

  • Characterize size and concentration using nanoparticle tracking analysis or dynamic light scattering

  • Perform functional assays to determine biological effects

What is the role of TSG101 in ESCRT-mediated processes and how can antibodies help elucidate these mechanisms?

TSG101 is a key component of the ESCRT-I complex with critical functions in cellular processes:

• ESCRT pathway functions: TSG101 is involved in:

  • Endosomal sorting of ubiquitinated cargo proteins

  • Multivesicular body (MVB) formation

  • Cytokinesis completion (requires interaction with CEP55)

  • Membrane receptor degradation

  • Viral budding

  • Exosome biogenesis

• Research applications using TSG101 antibodies:

  • Colocalization studies: Immunofluorescence with TSG101 antibodies combined with markers for endosomes, MVBs, and other ESCRT components can reveal spatial relationships during vesicle formation.

  • Protein-protein interactions: Immunoprecipitation with TSG101 antibodies can identify novel ESCRT pathway components and regulatory partners.

  • Functional studies: TSG101 antibodies can help monitor changes in ESCRT pathway components during cellular processes or disease states.

  • Mechanistic investigations: TSG101 overexpression can inhibit virus budding independently of its interaction with viral proteins, providing insights into ESCRT machinery function .

• Technical considerations:

  • Use monoclonal antibodies for highest specificity in colocalization studies

  • Polyclonal antibodies may provide stronger signals for protein detection

  • Consider epitope accessibility when studying protein complexes

How can TSG101 antibodies contribute to understanding the mechanisms of viral infection and potential therapeutic interventions?

TSG101 antibodies provide valuable tools for studying viral infection mechanisms:

• Virus-host interaction studies:

  • TSG101 interacts with the PTAPP motif in the p6 region of HIV-1 Gag protein

  • This interaction is essential for viral budding

  • TSG101 antibodies can be used in co-immunoprecipitation experiments to study these interactions

• Research applications:

  • Interaction mapping: Using TSG101 antibodies in immune capture assays has revealed that the interaction occurs through the PTAPP motif, as demonstrated by competition with PTAPP-containing peptides .

  • Functional domain analysis: TSG101 antibodies help identify which domains are critical for viral interactions, showing that the C-terminal half of the p6 region is not a TSG101-binding site .

  • Genetic variation studies: TSG101 antibodies can be used to study how different TSG101 haplotypes affect viral replication efficiency and disease progression .

• Therapeutic implications:

  • Identifying drugs that disrupt the TSG101-viral protein interaction

  • Developing peptide inhibitors based on PTAPP motifs

  • Targeting TSG101 domains that mediate viral budding

  • Understanding how genetic variants in TSG101 might affect therapeutic responses