Anti-RICTOR antibody [EPR22008]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-RICTOR antibody [EPR22008] (AB219950)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HCT 116 (human colorectal carcinoma cell line) cell line labeling RICTOR with ab219950 at 1/500 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells incubated with secondary antibody only) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-RICTOR antibody [EPR22008] (AB219950)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling RICTOR with ab219950 at 1/500 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells incubated with secondary antibody only) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

• WB

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Western blot - Anti-RICTOR antibody [EPR22008] (AB219950)

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

Exposure times : Lane 1 : 3 minutes; Lane 2 : 48 seconds; Lane 3-4 : 26 seconds.

All lanes:

Western blot - Anti-RICTOR antibody [EPR22008] (ab219950) at 1/1000 dilution

Lane 1:

HCT 116 (Human colorectal carcinoma cell line) whole cell lysate at 10 µg

Lane 2:

MDA-MB-231 (Human breast adenocarcinoma cell line) whole cell lysate at 10 µg

Lane 3:

SW480 (Human colorectal adenocarcinoma cell line) whole cell lysate at 10 µg

Lane 4:

PC-3 (Human prostate adenocarcinoma cell line) whole cell lysate at 10 µg

Secondary

Lane 1:

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 1/100000 dilution

Lanes 2 - 4:

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 1/50000 dilution

Predicted band size: 192 kDa

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• WB

Lab

Western blot - Anti-RICTOR antibody [EPR22008] (AB219950)

False colour image of Western blot : Anti-RICTOR antibody [EPR22008] staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab219950 was shown to bind specifically to RICTOR. A band was observed at 190 kDa in wild-type A549 cell lysates with no signal observed at this size in RICTOR knockout cell line ab277866 (knockout cell lysate ab288315). To generate this image, wild-type and RICTOR 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-RICTOR antibody [EPR22008] (ab219950) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

RICTOR knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human RICTOR knockout A549 cell line (<a href='/en-us/products/cell-lines/human-rictor-knockout-a549-cell-line-ab277866'>ab277866</a>)

Lane 3:

HeLa cell lysate at 20 µg

Predicted band size: 192 kDa

Observed band size: 190 kDa

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• WB

Unknown

Western blot - Anti-RICTOR antibody [EPR22008] (AB219950)

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

Exposure time : 26 seconds.

ab219950 was shown to specifically react with RICTOR in wild-type HAP1 cells as signal was lost in RICTOR knockout cells. Wild-type and RICTOR knockout samples were subjected to SDS-PAGE. ab219950 and ab181602 (Rabbit anti-GAPDH loading control) 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 1/50,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument using the ECL technique.

All lanes:

Western blot - Anti-RICTOR antibody [EPR22008] (ab219950) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

RICTOR knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 4:

HEK-293 (human epithelial cell line from embryonic kidney) 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 1/50000 dilution

Predicted band size: 192 kDa

false