TSG101 Antibody (Rabbit mAb) [K21M20]

Application: Reactivity: Human, Mouse, Rat

Lane 1: 293T, Lane 2: A431, Lane 3: Hela, Lane 4: MCF7

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human, Mouse, Rat

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
44 kDa

Positive Control	293T cells; A431 cells; HeLa cells; Jurkat cells; K-562 cells; MCF7 cells
Negative Control

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Datasheet & SDS

Biological Description

Specificity
TSG101 Antibody (Rabbit mAb) [K21M20] detects endogenous levels of total TSG101 protein.

Subcellular Location
Cytoplasm, Cytoskeleton, Endosome, Membrane, Nucleus

Uniprot ID
Q99816

Clone
K21M20

Synonym(s)
ESCRT-I complex subunit TSG101; TS101; TSG10; TSG101; tumor susceptibility 101; tumor susceptibility gene 10; tumor susceptibility gene 101; Tumor susceptibility gene 101 protein; tumor susceptibility protein; VPS23

Background
TSG101, the tumor susceptibility gene 101 protein, functions as a core subunit of ESCRT-I, the first-acting complex within the endosomal sorting complex required for transport machinery that governs sorting of ubiquitinated cargo through the endosomal compartment. The protein's N-terminal domain adopts a structural fold homologous to ubiquitin-conjugating E2 enzymes but lacks catalytic activity, since the active-site cysteine found in true E2 enzymes is replaced by a tyrosine residue, earning this region the designation ubiquitin E2 variant, or UEV, domain; the UEV domain also carries an additional N-terminal helix and an extended beta-hairpin not present in canonical E2 enzymes, while lacking the two C-terminal helices those enzymes typically contain. This UEV domain contains two functionally distinct binding pockets: one recognizes ubiquitin, and the other engages short proline-containing PT/SAP or PTAP tetrapeptide motifs, and structural analysis of the UEV domain bound to the PTAP peptide from HIV-1 p6 Gag shows the peptide docking into a bifurcated groove positioned directly above the vestigial, catalytically dead enzyme active site, with the alanine-proline dipeptide inserting into a deep pocket structurally reminiscent of the proline-recognition pockets found in SH3 and WW domains. Through PTAP motif recognition, TSG101 is recruited to sites where cargo requires sorting, and this same recruitment mechanism is exploited by HIV-1, whose Gag polyprotein carries a PTAP late-domain motif that binds the UEV domain and recruits TSG101, together with downstream ESCRT machinery, to the plasma membrane site of viral particle assembly; depleting cellular TSG101 arrests HIV-1 budding at a late stage, and viral budding is restored upon TSG101 reintroduction, directly linking this single protein-protein interaction to a complete step in the viral replication cycle. Beyond PTAP recognition, the ubiquitin-binding pocket of the UEV domain performs a distinct chaperone function during the early stages of virus particle assembly, since pharmacological disruption of ubiquitin binding specifically arrests assembly at an initiation step that is mechanistically separate from the later PTAP-dependent budding step, indicating that TSG101's two UEV binding pockets support sequential, non-redundant stages of the same overall process. TSG101 additionally participates in general cell proliferation control and in exosome biogenesis, and complete loss of TSG101 expression causes embryonic lethality through cell cycle arrest, underscoring that expression levels are tightly regulated rather than dispensable. Despite early characterization as a tumor suppressor, TSG101 expression is elevated in multiple human cancers, and the same UEV pockets that mediate PTAP and ubiquitin recognition during ESCRT-dependent trafficking and viral budding represent structurally defined, druggable surfaces now being explored for broad-spectrum antiviral development.

Background

TSG101, the tumor susceptibility gene 101 protein, functions as a core subunit of ESCRT-I, the first-acting complex within the endosomal sorting complex required for transport machinery that governs sorting of ubiquitinated cargo through the endosomal compartment. The protein's N-terminal domain adopts a structural fold homologous to ubiquitin-conjugating E2 enzymes but lacks catalytic activity, since the active-site cysteine found in true E2 enzymes is replaced by a tyrosine residue, earning this region the designation ubiquitin E2 variant, or UEV, domain; the UEV domain also carries an additional N-terminal helix and an extended beta-hairpin not present in canonical E2 enzymes, while lacking the two C-terminal helices those enzymes typically contain. This UEV domain contains two functionally distinct binding pockets: one recognizes ubiquitin, and the other engages short proline-containing PT/SAP or PTAP tetrapeptide motifs, and structural analysis of the UEV domain bound to the PTAP peptide from HIV-1 p6 Gag shows the peptide docking into a bifurcated groove positioned directly above the vestigial, catalytically dead enzyme active site, with the alanine-proline dipeptide inserting into a deep pocket structurally reminiscent of the proline-recognition pockets found in SH3 and WW domains. Through PTAP motif recognition, TSG101 is recruited to sites where cargo requires sorting, and this same recruitment mechanism is exploited by HIV-1, whose Gag polyprotein carries a PTAP late-domain motif that binds the UEV domain and recruits TSG101, together with downstream ESCRT machinery, to the plasma membrane site of viral particle assembly; depleting cellular TSG101 arrests HIV-1 budding at a late stage, and viral budding is restored upon TSG101 reintroduction, directly linking this single protein-protein interaction to a complete step in the viral replication cycle. Beyond PTAP recognition, the ubiquitin-binding pocket of the UEV domain performs a distinct chaperone function during the early stages of virus particle assembly, since pharmacological disruption of ubiquitin binding specifically arrests assembly at an initiation step that is mechanistically separate from the later PTAP-dependent budding step, indicating that TSG101's two UEV binding pockets support sequential, non-redundant stages of the same overall process. TSG101 additionally participates in general cell proliferation control and in exosome biogenesis, and complete loss of TSG101 expression causes embryonic lethality through cell cycle arrest, underscoring that expression levels are tightly regulated rather than dispensable. Despite early characterization as a tumor suppressor, TSG101 expression is elevated in multiple human cancers, and the same UEV pockets that mediate PTAP and ubiquitin recognition during ESCRT-dependent trafficking and viral budding represent structurally defined, druggable surfaces now being explored for broad-spectrum antiviral development.

References
https://pubmed.ncbi.nlm.nih.gov/11595185/ https://pubmed.ncbi.nlm.nih.gov/12379843/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%