Anti-Tuberin antibody [EPR22886-235]

• IP

Unknown

Immunoprecipitation - Anti-Tuberin antibody [EPR22886-235] (AB255612)

Tuberin was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg with ab255612 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab255612. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

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

Lane 2 : ab255612 IP in HeLa whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab255612 in HeLa whole cell lysate.

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

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-Tuberin antibody [EPR22886-235] (ab255612)

Predicted band size: 201 kDa

Observed band size: 200 kDa

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• WB

Lab

Western blot - Anti-Tuberin antibody [EPR22886-235] (AB255612)

Western blot : Anti-TSC2 antibody [EPR22886-235] (ab255612) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab255612 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 [EPR22886-235] (ab255612) at 1/1000 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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• WB

Lab

Western blot - Anti-Tuberin antibody [EPR22886-235] (AB255612)

The molecular weight observed is consistent with what has been described in the literature (PMID : 30774414; 10335945).

ab255612 was shown to specifically react with Tuberin in wild-type HAP1 cells as signal was lost in Tuberin knockout cells. Wild-type and Tuberin knockout samples were subjected to SDS-PAGE. ab255612 and ab129002 (Recombinant Anti-Vinculin antibody [EPR8185]) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/20,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc^™ MP instrument using the ECL technique.

Blocking and dilution buffer : 5% NFDM/TBST.

Exposire times.

Lanes 1-3 : 81 seconds; Lane 4 : 3 minutes.

All lanes:

Western blot - Anti-Tuberin antibody [EPR22886-235] (ab255612) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Tuberin knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 4:

HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 5:

A549 (human lung carcinoma epithelial cell), whole cell lysate at 20 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

false

• WB

Lab

Western blot - Anti-Tuberin antibody [EPR22886-235] (AB255612)

Blocking and dilution buffer : 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID : 30774414; 10335945).

This blot was developed using a higher sensitivity ECL substrate.

All lanes:

Western blot - Anti-Tuberin antibody [EPR22886-235] (ab255612) at 1/1000 dilution

Lane 1:

Human testis tissue lysate at 20 µg

Lane 2:

C2C12 (mouse myoblasts myoblast), whole cell lysate at 20 µg

Secondary

Lane 1:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Lane 2:

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

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-Tuberin antibody [EPR22886-235] (AB255612)

Blocking and dilution buffer : 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID : 30774414; 10335945).

All lanes:

Western blot - Anti-Tuberin antibody [EPR22886-235] (ab255612) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 20 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

false

Exposure time: 3min