Anti-PKR antibody [EPR19374]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PKR antibody [EPR19374] (AB184257)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilised bEnd.3 (mouse brain endothelial cell) cells labeling PKR with ab184257 at 1/500 dilution (red) compared with a Rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PKR antibody [EPR19374] (AB184257)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A549 (human lung carcinoma epithelial cell) labeling PKR with ab184257 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preabsorbed (ab150081) secondary antibody at 1/1000 dilution (green). Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 was used as a counterstain The nuclear counterstain is DAPI (blue) Confocal image showing cytoplasmic and weak nuclear staining in A549 cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PKR antibody [EPR19374] (AB184257)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized bEnd.3 (mouse brain endothelial cell) labeling PKR with ab184257 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preabsorbed (ab150081) secondary antibody at 1/1000 dilution (green). Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 was used as a counterstain The nuclear counterstain is DAPI (blue) Confocal image showing cytoplasmic and weak nuclear staining in bEnd.3 cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

• IP

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Immunoprecipitation - Anti-PKR antibody [EPR19374] (AB184257)

PKR was immunoprecipitated from mouse hippocampus lysate with ab184257 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed from the immunoprecipitate using ab184257 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution. Lane 1 : Mouse hippocampus tissue lysate 10 μg (Input). Lane 2 : ab184257 IP in Mouse hippocampus tissue lysate. Lane 3 : Rabbit IgG,monoclonal [EPR19374] - Isotype Control (ab172730) instead of ab184257 in mouse hippocampus tissue lysate. Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 31 seconds.

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• WB

Lab

Western blot - Anti-PKR antibody [EPR19374] (AB184257)

Lanes 1-3 : Merged signal (red and green). Green - ab184257 observed at 70 kDa. Red - loading control ab8245 observed at 36 kDa.

ab184257 Anti-PKR antibody [EPR19374] was shown to specifically react with PKR in wild-type A549 cells. Loss of signal was observed when knockout cell line ab267000 (knockout cell lysate ab256901) was used. Wild-type and PKR knockout samples were subjected to SDS-PAGE. ab184257 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PKR antibody [EPR19374] (ab184257) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

EIF2AK2 knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human EIF2AK2 (PKR) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-eif2ak2-pkr-knockout-a549-cell-line-ab267000'>ab267000</a>)

Lane 3:

K-562 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 62 kDa,86 kDa

Observed band size: 70 kDa,86 kDa

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• WB

Supplier Data

Western blot - Anti-PKR antibody [EPR19374] (AB184257)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PKR antibody [EPR19374] (ab184257) at 1/2000 dilution

Lane 1:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 2:

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

Lane 3:

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

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 62 kDa

Observed band size: 58 kDa

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Exposure time: 3min

• WB

Lab

Western blot - Anti-PKR antibody [EPR19374] (AB184257)

Lanes 1-5 : Merged signal (red and green). Green - ab184257 observed at 70 kDa. Red - loading control ab8245 observed at 36 kDa.

ab184257 Anti-PKR antibody [EPR19374] was shown to specifically react with PKR in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261824 (knockout cell lysate ab256899) was used. Wild-type and PKR knockout samples were subjected to SDS-PAGE. ab184257 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PKR antibody [EPR19374] (ab184257) at 1/1000 dilution

Lane 1:

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

Lane 2:

EIF2AK2 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

Western blot - Human EIF2AK2 (PKR) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-eif2ak2-pkr-knockout-hela-cell-line-ab261824'>ab261824</a>)

Lane 3:

Wild-type A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

EIF2AK2 knockout A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

Lane 5:

K562 (Human chronic myelogenous leukemia lymphoblast cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 62 kDa

Observed band size: 70 kDa

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• WB

Lab

Western blot - Anti-PKR antibody [EPR19374] (AB184257)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : EIF2AK2 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : K562 whole cell lysate (20 μg)
Lane 4 : HepG2 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab184257 observed at 70 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab184257 was shown to specifically react with EIF2AK2 when EIF2AK2 knockout samples were used. Wild-type and EIF2AK2 knockout samples were subjected to SDS-PAGE. ab184257 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1000 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-PKR antibody [EPR19374] (ab184257)

Predicted band size: 62 kDa

false

• WB

Supplier Data

Western blot - Anti-PKR antibody [EPR19374] (AB184257)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-PKR antibody [EPR19374] (ab184257) at 1/1000 dilution

All lanes:

bEnd.3 (mouse brain endothelial cell) 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: 62 kDa

false

Exposure time: 114s

• WB

Lab

Western blot - Anti-PKR antibody [EPR19374] (AB184257)

Blocking and diluting buffer : 5% NFDM/TBST

Lanes 1 - 2:

Western blot - Anti-CGA/hCG alpha antibody [EP3373] (<a href='/en-us/products/primary-antibodies/cga-hcg-alpha-antibody-ep3373-ab92738'>ab92738</a>) at 1/2000 dilution

Lane 1:

Western blot - Anti-PKR antibody [EPR19374] (ab184257) at 1/1000 dilution

Lane 1:

JAR (Human placenta choriocarcinoma epithelial cell) whole cell lysate

Lane 1:

bEnd.3 (mouse brain endothelial cell) whole cell lysate at 20 µg

Lane 2:

JAR (Human placenta choriocarcinoma epithelial cell) whole cell lysate treated with Hydroxyurea (10mM 24h)

Secondary

Lanes 1 - 2:

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

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

Observed band size: 18 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-PKR antibody [EPR19374] (AB184257)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 and 2 : 3 minutes; Lane 3,4 and 5 : 5 seconds.

All lanes:

Western blot - Anti-PKR antibody [EPR19374] (ab184257) at 1/2000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse heart lysate at 10 µg

Lane 3:

Rat brain lysate at 10 µg

Lane 4:

Rat heart lysate at 10 µg

Lane 5:

Rat spleen lysate at 10 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: 62 kDa

Observed band size: 58 kDa

false

• WB

Supplier Data

Western blot - Anti-PKR antibody [EPR19374] (AB184257)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1,2,3 and 4 : 3 minutes; Lane 5 : 30 seconds; Lane 6,7 and 8 : 10 seconds.

All lanes:

Western blot - Anti-PKR antibody [EPR19374] (ab184257) at 1/2000 dilution

Lane 1:

Mouse brain lysate at 20 µg

Lane 2:

Mouse cerebral cortex lysate at 20 µg

Lane 3:

Mouse hippocampus lysate at 20 µg

Lane 4:

Mouse lung lysate at 20 µg

Lane 5:

Mouse thymus lysate at 20 µg

Lane 6:

Rat brain lysate at 20 µg

Lane 7:

Rat cerebral cortex lysate at 20 µg

Lane 8:

4T1 (Mouse mammary gland carcinoma 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: 62 kDa

false