Anti-Glutamine Synthetase antibody [EPR16661]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glutamine Synthetase antibody [EPR16661] (AB197024)

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.

• WB

Supplier Data

Western blot - Anti-Glutamine Synthetase antibody [EPR16661] (AB197024)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Glutamine Synthetase antibody [EPR16661] (ab197024) at 1/1000 dilution

All lanes:

Human glioma lysate at 10 µg

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

false

Exposure time: 1min

• WB

Lab

Western blot - Anti-Glutamine Synthetase antibody [EPR16661] (AB197024)

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:

Western blot - Anti-Glutamine Synthetase antibody [EPR16661] (ab197024)

Predicted band size: 42 kDa

false

• WB

Lab

Western blot - Anti-Glutamine Synthetase antibody [EPR16661] (AB197024)

Western blot : Anti-Glutamine Synthetase antibody [EPR16661] ab197024 staining at 1/1000 dilution, shown in green; Mouse anti-CANX ab238078 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 42 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in GLUL knockout U-87 MG cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5% Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Glutamine Synthetase antibody [EPR16661] (ab197024) at 1/1000 dilution

Lane 1:

Wild-type U-87 MG at 20 µg

Lane 2:

GLUL knockout U-87 MG at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 42 kDa

Observed band size: 42 kDa

false

• WB

Lab

Western blot - Anti-Glutamine Synthetase antibody [EPR16661] (AB197024)

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.

All lanes:

Western blot - Anti-Glutamine Synthetase antibody [EPR16661] (ab197024) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 40 µg

Lane 2:

GLUL knockout HeLa cell lysate at 40 µg

Predicted band size: 42 kDa

Observed band size: 42 kDa

false

• WB

Supplier Data

Western blot - Anti-Glutamine Synthetase antibody [EPR16661] (AB197024)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Glutamine Synthetase antibody [EPR16661] (ab197024) at 1/1000 dilution

Lane 1:

Human fetal liver lysate at 20 µg

Lane 2:

HeLa (Human epithelial cells from cervix adenocarcinoma) lysate at 20 µg

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

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-Glutamine Synthetase antibody [EPR16661] (AB197024)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Glutamine Synthetase antibody [EPR16661] (ab197024) at 1/1000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse spleen lysate at 10 µg

Lane 3:

NIH/3T3 (Mouse embryo fibroblast cells) lysate at 10 µg

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

false

Exposure time: 3min