Anti-S6K1 antibody [E175]

• WB

Lab

Western blot - Anti-S6K1 antibody [E175] (AB32359)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : S6K1 knockout HAP1 cell lysate (20 μg)
Lane 3 : MCF7 cell lysate (20 μg)
Lane 4 : HEK293 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab32359 observed at 68 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab32359 was shown to recognize S6K1 when S6K1 knockout samples were used, along with additional cross-reactive bands. Wild-type and S6K1 knockout samples were subjected to SDS-PAGE. ab32359 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

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Western blot - Anti-S6K1 antibody [E175] (ab32359)

Predicted band size: 59 kDa

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• WB

Unknown

Western blot - Anti-S6K1 antibody [E175] (AB32359)

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Western blot - Anti-S6K1 antibody [E175] (ab32359)

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HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate

Predicted band size: 59 kDa

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• WB

Lab

Western blot - Anti-S6K1 antibody [E175] (AB32359)

Western blot : Rabbit Monoclonal[E175] to S6K1 ab32359 staining at 1/5000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 60 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in RPS6KB1 knockout U-87 MG cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-S6K1 antibody [E175] (ab32359) at 1/5000 dilution

Lane 1:

Wild-type U-87 MG at 20 µg

Lane 2:

RPS6KB1 knockout U-87 MG at 20 µg

Secondary

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Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 59 kDa

Observed band size: 60 kDa

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• WB

Lab

Western blot - Anti-S6K1 antibody [E175] (AB32359)

Blocking and dilution buffer and concentration : 5% NFDM/TBST. ab205606 was also used at 1/1000 dilution to detect C-Myc/DDK tagged human S6K1 and S6K2 recombinant proteins shown on the right panel. ab32539 showed cross reactivity with S6K2 in recombinant protein testing. S6K1 and S6K2 have different observed band size in endogenous and recombinant protein tests.

Lanes 1 - 2:

Western blot - Anti-S6K1 antibody [E175] (ab32359) at 1/1000 dilution

Lanes 1 - 2:

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (<a href='/en-us/products/primary-antibodies/ddddk-tag-binds-to-flag-tag-sequence-antibody-epr20018-251-ab205606'>ab205606</a>) at 1/1000 dilution

Lane 1:

C-Myc/DDK tagged human S6K1 recombinant protein at 10 ng

Lane 2:

C-Myc/DDK tagged human S6K2 recombinant protein at 10 ng

Secondary

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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: 59 kDa

Observed band size: 75 kDa,60 kDa

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Exposure time: 10s