Anti-GTPase HRAS antibody [Y132]

• IP

Unknown

Immunoprecipitation - Anti-GTPase HRAS antibody [Y132] (AB32417)

ab32417 (purified) at 1/60 immunoprecipitating GTPase in 10 μg mouse brain whole cell lysate (Lanes 1 and 2, observed at 21 kDa). Lane 3 - PBS. For western blotting, HRP Veriblot for IP Detection Reagent (ab131366) was used for detection (1/10 000). Blocking buffer and concentration : 5% NFDM/TBST Dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-GTPase HRAS antibody [Y132] (ab32417)

Predicted band size: 21 kDa

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• WB

Lab

Western blot - Anti-GTPase HRAS antibody [Y132] (AB32417)

Western blot : Anti-GTPase HRAS antibody [Y132] ab32417 staining at 1/1000 dilution, shown in green; Mouse anti-CANX ab238078 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 22 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in HRAS knockout MCF7 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-GTPase HRAS antibody [Y132] (ab32417) at 1/1000 dilution

Lane 1:

Wild-type MCF7 whole cell lysate at 20 µg

Lane 2:

HRAS knockout MCF7 whole cell lysate at 20 µg

Lane 3:

HEK-293 whole cell lysate at 20 µg

Lane 4:

HeLa whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 21 kDa

Observed band size: 22 kDa

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• WB

Lab

Western blot - Anti-GTPase HRAS antibody [Y132] (AB32417)

Blocking buffer : 5% NFDM/TBST
Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-GTPase HRAS antibody [Y132] (ab32417) at 1/1000 dilution

Lane 1:

MCF7 cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

Predicted band size: 21 kDa

Observed band size: 21 kDa

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• WB

Lab

Western blot - Anti-GTPase HRAS antibody [Y132] (AB32417)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32417 observed at 21 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32417 was shown to recognize HRAS in wild-type HEK-293 cells as signal was lost at the expected MW in HRAS knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and HRAS knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. ab32417 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/500 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-GTPase HRAS antibody [Y132] (ab32417) at 1/500 dilution

Lane 1:

Wild-type HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

HRAS knockout HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

Western blot - Human HRAS knockout HEK-293 cell line (<a href='/en-us/products/cell-lines/human-hras-knockout-hek-293-cell-line-ab260858'>ab260858</a>)

Lane 3:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 4:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Predicted band size: 21 kDa

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• WB

Unknown

Western blot - Anti-GTPase HRAS antibody [Y132] (AB32417)

All lanes:

Western blot - Anti-GTPase HRAS antibody [Y132] (ab32417) at 1/500 dilution

Lane 1:

MCF7 cell lysate

Lane 2:

PC12 cell lysate

Predicted band size: 21 kDa

Observed band size: 21 kDa

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• WB

Lab

Western blot - Anti-GTPase HRAS antibody [Y132] (AB32417)

Blocking buffer : 5% NFDM/TBST
Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-GTPase HRAS antibody [Y132] (ab32417) at 1/2500 dilution

Lane 1:

mouse brain lysate at 10 µg

Lane 2:

rat brain lysate at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

Predicted band size: 21 kDa

Observed band size: 21 kDa

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