All lanes : Anti-PKR antibody [EPR19374] (ab184257) at 1/1000 dilution

Lane 1 : Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : EIF2AK2 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : Wild-type A549 (Human lung carcinoma cell line) whole cell lysate
Lane 4 : EIF2AK2 knockout A549 (Human lung carcinoma cell line) whole cell lysate
Lane 5 : K562 (Human chronic myelogenous leukemia lymphoblast cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 58 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?

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.

Flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling PKR with ab184257 at 1/120 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 (FITC) at 1/500 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling PKR with ab184257 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasm and weakly nuclear staining on HeLa cell line. 

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [EPR19374] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab184257 at 1/100 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2: ab7291  at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 Mouse embryonic fibroblast cell line) cells labeling PKR with ab184257 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasm and weakly nuclear staining on NIH/3T3 cell line. 

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [EPR19374]- Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab184257 at 1/100 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2: ab7291  at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

All lanes : Anti-PKR antibody [EPR19374] (ab184257) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : EIF2AK2 knockout A549 cell lysate
Lane 3 : K-562 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 58 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?

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

Lane 1 : Wild-type A549 cell lysate
Lane 3 : K-562 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 58 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?

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 ab266999 (knockout cell lysate ab256900) 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.

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: EIF2AK2 knockout HAP1 whole cell lysate (20 µg)
Lane 3: K652 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 : Anti-PKR antibody [EPR19374] (ab184257) at 1/2000 dilution

Lane 1 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 58 kDa
Observed band size: 58 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PKR antibody [EPR19374] (ab184257) at 1/2000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse cerebral cortex lysate
Lane 3 : Mouse hippocampus lysate
Lane 4 : Mouse lung lysate
Lane 5 : Mouse thymus lysate
Lane 6 : Rat brain lysate
Lane 7 : Rat cerebral cortex lysate
Lane 8 : 4T1 (Mouse mammary gland carcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 58 kDa

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

Lane 1 : Mouse brain lysate
Lane 2 : Mouse heart lysate
Lane 3 : Rat brain lysate
Lane 4 : Rat heart lysate
Lane 5 : Rat spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 58 kDa
Observed band size: 58 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

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

All lanes : Anti-PKR antibody [EPR19374] (ab184257) at 1/2000 dilution

Lane 1 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 2 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 4 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 58 kDa
Observed band size: 58 kDa


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID:12892903).

PKR was immunoprecipitated from 1mg of NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate with ab184257 at 1/40 dilution.

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 1/10000 dilution.

Lane 1: NIH/3T3 whole cell lysate 10µg (Input).

Lane 2: ab184257 IP in NIH/3T3 whole cell lysate.

Lane 3: Rabbit IgG,monoclonal [EPR19374] - Isotype Control (ab172730) instead of ab184257 in NIH/3T3 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 10 seconds.