Anti-GCN2 antibody [EPR5970(2)]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GCN2 antibody [EPR5970(2)] (AB134053)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GCN2 antibody [EPR5970(2)] (AB134053)

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 Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-GCN2 antibody [EPR5970(2)] (AB134053)

Intracellular 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GCN2 antibody [EPR5970(2)] (AB134053)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GCN2 antibody [EPR5970(2)] (AB134053)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-GCN2 antibody [EPR5970(2)] (AB134053)

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.

• WB

Lab

Western blot - Anti-GCN2 antibody [EPR5970(2)] (AB134053)

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.

All lanes:

Western blot - Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

EIF2AK4 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human EIF2AK4 (GCN2) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-eif2ak4-gcn2-knockout-hek-293t-cell-line-ab267246'>ab267246</a>)

Predicted band size: 187 kDa

Observed band size: 187 kDa

false

• WB

Lab

Western blot - Anti-GCN2 antibody [EPR5970(2)] (AB134053)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

ab134053 is more sensitive than ab302609 in WB testing.

Lanes 1 - 2:

Western blot - Anti-GCN2 antibody [EPR25345-125] (<a href='/en-us/products/primary-antibodies/gcn2-antibody-epr25345-125-ab302609'>ab302609</a>) at 1/1000 dilution

Lanes 3 - 4:

Western blot - Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/1000 dilution

Lanes 1 and 3:

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

Lanes 2 and 4:

293T (Human epithelial cell line from embryonic kidney) 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 1/20000 dilution

Observed band size: 187 kDa

false

Exposure time: 60s

• WB

Lab

Western blot - Anti-GCN2 antibody [EPR5970(2)] (AB134053)

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.

All lanes:

Western blot - Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

EIF2AK4 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human EIF2AK4 (GCN2) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-eif2ak4-gcn2-knockout-hek-293t-cell-line-ab267247'>ab267247</a>)

Predicted band size: 112 kDa,13 kDa,187 kDa,55 kDa,69 kDa

Observed band size: 187 kDa

false

• WB

Lab

Western blot - Anti-GCN2 antibody [EPR5970(2)] (AB134053)

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:

Western blot - Anti-GCN2 antibody [EPR5970(2)] (ab134053)

Predicted band size: 187 kDa,55 kDa

false

• WB

Unknown

Western blot - Anti-GCN2 antibody [EPR5970(2)] (AB134053)

All lanes:

Western blot - Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

293T cell lysate at 10 µg

Lane 3:

MOLT4 cell lysate at 10 µg

Lane 4:

A549 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 187 kDa

false

• WB

Lab

Western blot - Anti-GCN2 antibody [EPR5970(2)] (AB134053)

Blocking and diluting buffer : 5% NFDM /TBST.

All lanes:

Western blot - Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/50000 dilution

All lanes:

MOLT-4 (Human lymphoblastic leukemia 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 1/20000 dilution

Predicted band size: 187 kDa

Observed band size: 187 kDa

false

• WB

Lab

Western blot - Anti-GCN2 antibody [EPR5970(2)] (AB134053)

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/10000 dilution

Lane 1:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 15 µg

Lane 2:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 15 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: 187 kDa

Observed band size: 187 kDa

false