Anti-PERK antibody [EPR19876-294]

• WB

Lab

Western blot - Anti-PERK antibody [EPR19876-294] (AB229912)

Blocking/Dilution buffer : 5% NFDM/TBST.

ab229912 was shown to specifically react with PERK in wild-type HAP1 cells as signal was lost in PERK knockout cells. Wild-type and PERK knockout samples were subjected to SDS-PAGE. ab229912 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 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument using the ECL technique.

This antibody also recognizes unidentifiable proteins below 75 kDa.

All lanes:

Western blot - Anti-PERK antibody [EPR19876-294] (ab229912) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

PERK knockout HAP1 whole cell lysate at 20 µg

Lane 3:

U-87 MG (human glioblastoma-astrocytoma epithelial cell line) whole cell lysate at 20 µg

Lane 4:

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

Lane 5:

HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 20 µg

Lane 6:

MCF7 (human breast adenocarcinoma cell line) 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 1/100000 dilution

Predicted band size: 125 kDa

Observed band size: 150 kDa

false

Exposure time: 125s

• WB

Lab

Western blot - Anti-PERK antibody [EPR19876-294] (AB229912)

Western blot : Rabbit Monoclonal[EPR19876-294] to PERK ab229912 staining at 1/1000 dilution, shown in green; Mouse anti-CANX (ab238078) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 140 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in EIF2AK3 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 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-PERK antibody [EPR19876-294] (ab229912) at 1/1000 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

Western blot - Human EIF2AK3 knockout MCF7 cell line (ab289377) at 20 µg

Lane 2:

EIF2AK3 knockout MCF7 at 20 µg

Lane 3:

Wild-type A549 ab288558 at 20 µg

Lane 4:

EIF2AK3 knockout A549 <a href='/en-us/products/cell-lines/human-eif2ak3-knockout-a549-cell-line-ab288905'>ab288905</a> at 20 µg

Secondary

All lanes:

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

Predicted band size: 125 kDa

Observed band size: 140 kDa

false

• WB

Lab

Western blot - Anti-PERK antibody [EPR19876-294] (AB229912)

Western blot : Anti-EIF2AK3 antibody [EPR19876-294] (ab229912) 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, ab229912 was shown to bind specifically to EIF2AK3. A band was observed at 145 kDa in wild-type A549 cell lysates with no signal observed at this size in EIF2AK3 knockout cell line. To generate this image, wild-type and EIF2AK3 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-PERK antibody [EPR19876-294] (ab229912) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

EIF2AK3 knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human EIF2AK3 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-eif2ak3-knockout-a549-cell-line-ab288905'>ab288905</a>)

Lane 3:

MCF7 cell lysate at 20 µg

Lane 4:

Human eye 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

Observed band size: 145 kDa

false

• WB

Lab

Western blot - Anti-PERK antibody [EPR19876-294] (AB229912)

Exposure time : Lanes 1-2 : 37 seconds; Lanes 3-6 : 3 minutes.

Blocking/Dilution buffer : 5% NFDM/TBST.

This antibody also recognizes unidentifiable proteins below 37 kDa.

All lanes:

Western blot - Anti-PERK antibody [EPR19876-294] (ab229912) at 1/1000 dilution

Lane 1:

RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (mouse embyro fibroblast cell line) whole cell lysate at 20 µg

Lane 3:

PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate at 20 µg

Lane 4:

Mouse brain lysate at 20 µg

Lane 5:

Rat brain lysate at 20 µg

Lane 6:

Rat liver 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 1/100000 dilution

Predicted band size: 125 kDa

Observed band size: 150 kDa

false