Anti-GCN2 antibody [EPR5970(2)] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GCN2 antibody [EPR5970(2)] - BSA and Azide free (AB157775)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134053).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-GCN2 antibody [EPR5970(2)] - BSA and Azide free (AB157775)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134053).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GCN2 antibody [EPR5970(2)] - BSA and Azide free (AB157775)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134053).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-GCN2 antibody [EPR5970(2)] - BSA and Azide free (AB157775)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134053).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GCN2 antibody [EPR5970(2)] - BSA and Azide free (AB157775)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134053).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GCN2 antibody [EPR5970(2)] - BSA and Azide free (AB157775)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134053).

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

• WB

Lab

Western blot - Anti-GCN2 antibody [EPR5970(2)] - BSA and Azide free (AB157775)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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)] (<a href='/en-us/products/primary-antibodies/gcn2-antibody-epr59702-ab134053'>ab134053</a>) 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)] - BSA and Azide free (AB157775)

This data was developed using the same antibody clone in a different buffer formation (ab134052).

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 (IRDye800^®CW) 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)] (<a href='/en-us/products/primary-antibodies/gcn2-antibody-epr59702-ab134053'>ab134053</a>) 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