重组Anti-PKR抗体[YE350] (ab32052)

Lanes 1-5: Merged signal (red and green). Green - ab32052 observed at 70 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab32052 Anti-PKR antibody [YE350] 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. ab32052 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.

Immunocytochemistry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling PKR with Purified ab32052 at 1:50 dilution (5.04 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor® 488,ab150077) was used as the secondary antibody at 1:1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Blocking buffer: 5% NFDM/TBST

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling PKR with Purified ab32052 at 1/30 dilution (10 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Purified ab32052 at 1/20 dilution (1µg) immunoprecipitating PKR in Jurkat whole cell lysate.
Lane 1 (input): Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10µg
Lane 2 (+): ab32052 + Jurkat whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32052 in Jurkat whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 68 kDa
Possible degradation bands are observed between 20-30kDa.

Lanes 1-3: Merged signal (red and green). Green - ab32052 observed at 70 kDa. Red - loading control ab8245 observed at 36 kDa. 

 ab32052 Anti-PKR antibody [YE350] 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.  ab32052 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.

Lanes 1-3: Merged signal (red and green). Green - ab32052 observed at 70 kDa. Red - loading control ab8245 observed at 36 kDa. 

 ab32052 Anti-PKR antibody [YE350] 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.  ab32052 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 - ab32052 observed at 70 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab32052 was shown to specifically react with EIF2AK2 when EIF2AK2 knockout samples were used. Wild-type and EIF2AK2 knockout samples were subjected to SDS-PAGE. Ab32052 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.