Human NRAS knockout HEK-293T cell lysate

• WB

Lab

Western blot - Human NRAS knockout HEK-293T cell lysate (AB258542)

Lane 1 : Wild-type HEK-293T cell lysate 20 μg
Lane 2 : NRAS knockout HEK-293T cell lysate 20 μg
Lane 3 : MCF7 cell lysate 20 μg
Lane 4 : A549 cell lysate 20 μg
Lanes 1 - 4 : Merged signal (red and green). Green - ab198820 observed at 22 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.
ab198820 was shown to react with NRAS in wild-type HEK-293T cells in Western blot with loss of signal observed in NRAS knockout cell line ab266684 (NRAS knockout cell lysate ab258542). Wild-type HEK-293T and NRAS knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab198820 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 ^°C at a 1 in 200 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-NRAS antibody - C-terminal (<a href='/en-us/products/primary-antibodies/nras-antibody-c-terminal-ab198820'>ab198820</a>) at 1/200 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

NRAS knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human NRAS knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-nras-knockout-hek-293t-cell-line-ab266684'>ab266684</a>)

Lane 3:

MCF7 cell lysate at 20 µg

Lane 4:

A549 cell lysate at 20 µg

Predicted band size: 21 kDa

Observed band size: 22 kDa

false

• WB

Lab

Western blot - Human NRAS knockout HEK-293T cell lysate (AB258542)

Lane 1 : Wild-type HEK-293T cell lysate 20 μg
Lane 2 : NRAS knockout HEK-293T cell lysate 20 μg
Lane 3 : MCF7 cell lysate 20 μg
Lane 4 : A549 cell lysate 20 μg
Lanes 1 - 4 : Merged signal (red and green). Green - ab167136 observed at 22 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.
ab167136 was shown to react with NRAS in wild-type HEK-293T cells in Western blot with loss of signal observed in NRAS knockout cell line ab266684 (NRAS knockout cell lysate ab258542). Wild-type HEK-293T and NRAS knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab167136 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 ^°C at 0.5 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-NRAS antibody (<a href='/en-us/products/primary-antibodies/nras-antibody-ab167136'>ab167136</a>) at 0.5 µg/mL

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

NRAS knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human NRAS knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-nras-knockout-hek-293t-cell-line-ab266684'>ab266684</a>)

Lane 3:

MCF7 cell lysate at 20 µg

Lane 4:

A549 cell lysate at 20 µg

Predicted band size: 21 kDa

Observed band size: 22 kDa

false

• WB

Lab

Western blot - Human NRAS knockout HEK-293T cell lysate (AB258542)

Lane 1 : Wild-type HEK-293T cell lysate 20 μg
Lane 2 : NRAS knockout HEK-293T cell lysate 20 μg
Lane 3 : MCF7 cell lysate 20 μg
Lane 4 : A549 cell lysate 20 μg
False colour image of Western blot : Anti-NRAS antibody staining at 0.5 μg/ml, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab167136 was shown to bind specifically to NRAS. A band was observed at 21 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in NRAS CRISPR-Cas9 edited cell line ab266684 (NRAS CRISPR-Cas9 edited cell lysate ab258542). The band observed in the CRISPR-Cas9 edited lysate lane above 21 kDa is likely to represent NRAS with an insertion. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and NRAS CRISPR-Cas9 edited HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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-NRAS antibody (<a href='/en-us/products/primary-antibodies/nras-antibody-ab167136'>ab167136</a>) at 0.5 µg/mL

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

NRAS knockout HEK-293T cell lysate at 20 µg

Lane 3:

MCF7 cell lysate at 20 µg

Lane 4:

A549 cell lysate at 20 µg

Predicted band size: 21 kDa

Observed band size: 21 kDa

false

• Sanger seq

Unknown

Sanger Sequencing - Human NRAS knockout HEK-293T cell lysate (AB258542)

Allele-2 : 5 bp deletion in exon 2

• Sanger seq

Unknown

Sanger Sequencing - Human NRAS knockout HEK-293T cell lysate (AB258542)

Allele-1 : 8 bp deletion in exon 2