Anti-Tuberin antibody [EP1107Y]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tuberin antibody [EP1107Y] (AB52936)

Immunohistochemical analysis of paraffin-embedded human adenocarcinoma of uterus using ab52936 at a dilution of 1/100-1/250.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Tuberin antibody [EP1107Y] (AB52936)

ab52936 stained HeLa cells. The cells were 4% formaldehyde fixed for 10 minutes at room temperature and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab52936 at 1/100 dilution) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43μM for 1hour at room temperature.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Tuberin antibody [EP1107Y] (AB52936)

Overlay histogram showing SH-SY5Y (Human neuroblastoma epithelial cell) cells stained with ab52936 (red line). The cells were fixed with 4% Paraformaldehydeand then permeabilized with 90% Methanol. The secondary antibody used was Alexa Fluorr^® 488 goat anti-rabbit IgG (H&L) ab150077 at 1/2000 dilution. Isotype control antibody (black line) was rabbit IgG (monoclonal). Unlabelled sample (blue line) was Cell without incubation with primary antibody and secondary antibody.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Tuberin antibody [EP1107Y] (AB52936)

Overlay histogram showing HeLa cells stained with ab52936 (red line). The cells were fixed with 80% methanol (5 min)/ and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab52936 1/10000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% formaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• WB

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Western blot - Anti-Tuberin antibody [EP1107Y] (AB52936)

The cell lysates was prepared in 1%SDS Hot lysis method.

All lanes:

Western blot - Anti-Tuberin antibody [EP1107Y] (ab52936) at 1/100000 dilution

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SH-SY5Y cell lysate at 10 µg

Secondary

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HRP-labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 201 kDa

Observed band size: ~200 kDa

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• WB

Lab

Western blot - Anti-Tuberin antibody [EP1107Y] (AB52936)

Blocking/Diluting Buffer and concentration :
5% NFDM/TBST

All lanes:

Western blot - Anti-Tuberin antibody [EP1107Y] (ab52936) at 1/20000 dilution

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HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate prepared in 1%SDS Hot lysis method at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 201 kDa

Observed band size: 200 kDa

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• IP

Lab

Immunoprecipitation - Anti-Tuberin antibody [EP1107Y] (AB52936)

Tuberin was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg with ab52936 at 1/50 dilution (2μg). VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg

Lane 2 : ab52936 IP in HeLa whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab52936 in HeLa whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Tuberin antibody [EP1107Y] (ab52936)

Predicted band size: 201 kDa

Observed band size: 200 kDa

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• WB

Supplier Data

Western blot - Anti-Tuberin antibody [EP1107Y] (AB52936)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : Tuberin knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab52936 observed at 180 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab52936 was shown to specifically react with tuberin in wild-type HAP1 cells. No band was observed when tuberin knockout samples were used. Wild-type and tuberin knockout samples were subjected to SDS-PAGE. ab52936 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Tuberin antibody [EP1107Y] (ab52936)

Predicted band size: 201 kDa

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• WB

Lab

Western blot - Anti-Tuberin antibody [EP1107Y] (AB52936)

Anti-TSC2 antibody [EP1107Y] (ab52936) staining at 1/20000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab52936 detected a band observed at 200 kDa in wild-type MCF7 cell lysates with no change observed in the TSC2 knockout cell line ab286525. To generate this image, wild-type and TSC2 knockout MCF7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Tuberin antibody [EP1107Y] (ab52936) at 1/20000 dilution

Lane 1:

Wild-type MCF7 cell lysate at 20 µg

Lane 2:

Western blot - Human TSC2 knockout MCF7 cell line (<a href='/en-us/products/cell-lines/human-tsc2-knockout-mcf7-cell-line-ab286525'>ab286525</a>)

Lane 2:

TSC2 knockout MCF7 cell lysate at 20 µg

Lane 3:

Wild-type HAP1 cell lysate at 20 µg

Lane 4:

TSC2 knockout HAP1 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 201 kDa

Observed band size: 200 kDa

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• WB

Lab

Western blot - Anti-Tuberin antibody [EP1107Y] (AB52936)

Western blot : Anti-TSC2 antibody [EP1107Y] (ab52936) staining at 1/50000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab52936 was shown to bind specifically to TSC2. A band was observed at 200 kDa in wild-type A549 cell lysates with no signal observed at this size in TSC2 knockout cell line. To generate this image, wild-type and TSC2 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Tuberin antibody [EP1107Y] (ab52936) at 1/50000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

TSC2 knockout A549 cell lysate at 20 µg

Lane 3:

Wild-type HAP1 cell lysate at 20 µg

Lane 4:

TSC2 knockout HAP1 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

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