All lanes : Anti-Glutamine Synthetase antibody [EPR16661] (ab197024) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : GLUL knockout HeLa cell lysate

Lysates/proteins at 40 µg per lane.

Performed under reducing conditions.

Predicted band size: 42 kDa
Observed band size: 42 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab197024 observed at 42 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab197024 was shown to react with Glutamine Synthetase in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261737 (knockout cell lysate ab256930) was used. Wild-type HeLa and GLUL knockout HeLa cell lysates were subjected to SDS-PAGE. ab197024 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 whole cell lysate (20 µg)
Lane 2: Glutamine Synthetase knockout HAP1 whole cell lysate (20 µg)
Lane 3: Human brain whole cell lysate (20 µg)
Lane 4: A431 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab197024 observed at 42 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab197024 was shown to specifically react with Glutamine Synthetase in wild-type HAP1 cells as signal was lost in Glutamine Synthetase knockout cells. Wild-type and Glutamine Synthetase knockout samples were subjected to SDS-PAGE. ab197024 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 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-Glutamine Synthetase antibody [EPR16661] (ab197024) at 1/1000 dilution

Lane 1 : Human fetal liver lysate
Lane 2 : HeLa (Human epithelial cells from cervix adenocarcinoma) lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 42 kDa
Observed band size: 42 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-Glutamine Synthetase antibody [EPR16661] (ab197024) at 1/1000 dilution + Human glioma lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 42 kDa
Observed band size: 42 kDa


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Glutamine Synthetase antibody [EPR16661] (ab197024) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse spleen lysate
Lane 3 : NIH/3T3 (Mouse embryo fibroblast cells) lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 42 kDa
Observed band size: 42 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human hepatocellular carcinoma tissue labeling Glutamine Synthetase with ab197024 at 1/8000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Cytoplasm staining on Human hepatocellular carcinoma tissue is observed. Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.