All lanes : Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : EIF2AK4 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 187 kDa
Observed band size: 187 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab134053 observed at 187 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab134053 was shown to react with GCN2 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab267247 (knockout cell lysate ab256903) was used. Wild-type HEK-293T and EIF2AK4 knockout HEK-293T cell lysates were subjected to SDS-PAGE. ab134053 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 1000 dilution and a 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/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma cell line) cells labeling GCN2  with ab134053 at 1/250 dilution (4.0μg/ml). The cells were co-stained with ab195889, an Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 (2.5 μg/ml). Cells were fixed with 100% methanol. ab150077, a Goat anti-rabbit IgG(Alexa Fluor^® 488) secondary antibody was used at 1/1000 dilution. DAPI was used as the nuclear counter stain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carconoma tissue sections labeling GCN2 with purified ab134053 at 1/100 dilution (10 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, PH9. ab97051, a Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1/500 dilution. Tissue was counterstained with hematoxylin. PBS instead of the primary antibody was used as the negative control.

Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma cell line) cells labeling GCN2 with purified ab134053 at 1/100 dilution (10 ug/ml). Cells were fixed with 4% paraformaldehyde. A Goat anti-rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1/2000 dilution. Rabbit monoclonal IgG (Black) was used as the isotypre control. Cells without incubation with the primary antibody and secondary antibody (Blue) is the unlabeled control.

All lanes : Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : EIF2AK4 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 187 kDa
Observed band size: 187 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab134053 observed at 187 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab134053 was shown to react with GCN2 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab267246 (knockout cell lysate ab256902) was used. Wild-type HEK-293T and EIF2AK4 knockout HEK-293T cell lysates were subjected to SDS-PAGE. ab134053 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 1000 Dilution and a 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 cell lysate (20 µg)
Lane 2: GCN2 knockout HAP1 cell lysate (20 µg)
Lane 3: MOLT4 cell lysate (20 µg)
Lane 4: A549 cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab134053 observed at 171 kDa. Red - loading control, ab18058, observed at 124 kDa.

Unpurified ab134053 was shown to recognize GCN2 when GCN2 knockout samples were used, along with additional cross-reactive bands. Wild-type and GCN2 knockout samples were subjected to SDS-PAGE. ab134053 and ab18058 (loading control to Vinculin) were diluted at 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. 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 1/10000 dilution for 1 h at room temperature before imaging.

All lanes : Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/10000 dilution (purified)

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate

Lysates/proteins at 15 µg per lane.

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

Predicted band size: 187 kDa
Observed band size: 187 kDa

Blocking and diluting buffer: 5% NFDM/TBST.

Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/50000 dilution (purified) + MOLT-4 (Human lymphoblastic leukemia cell line) whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 187 kDa
Observed band size: 187 kDa

Blocking and diluting buffer: 5% NFDM /TBST.

All lanes : Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/1000 dilution (Unpurified)

Lane 1 : HeLa cell lysate
Lane 2 : 293T cell lysate
Lane 3 : MOLT4 cell lysate
Lane 4 : A549 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 187 kDa

Immunofluorescence staining of MCF-7 cells with purified ab134053 at a working dilution of 1/500, counter-stained with DAPI. The secondary antibody was an Alexa Fluor^® 488 conjugated goat anti-rabbit (ab150077), used at a dilution of 1/1000. The cells were fixed in 100% methanol. The negative control is shown in bottom right hand panel - for the negative control, PBS was used instead of the primary antibody.

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labelling GCN2 with unpurified ab134053 at 1/100 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Overlay histogram showing HeLa cells stained with unpurified ab134053 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab134053, 1/10000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.