Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebral cortex tissue labelling Glutaminase with purified ab156876 at a dilution of 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Lane 1: Wild type HAP1 whole cell lysate (20 µg) Lane 2: GLS knockout HAP1 whole cell lysate (20 µg) Lane 3: HeLa whole cell lysate (20 µg) Lane 4: HEK293 whole cell lysate (20 µg) Lanes 1 - 4: Merged signal (red and green). Green - ab156876 observed at 62 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab156876 was shown to recognize GLS in wild-type HAP1 cells as signal was lost at the expected MW in HAP1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and GLS knockout samples were subjected to SDS-PAGE. Ab156876 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10,000 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 1/10,000 dilution for 1hr at room temperature before imaging.

Blue arrow corresponds to isoform 1 KGA.

Red arrow corresponds to isoform 3 GAC.

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Glutaminase with purified ab156876 at 1/250. Cells were fixed with 100% methanol and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/250) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

Flow cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Glutaminase (red) with ab156876 at a 1/400 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies.

All lanes : Anti-Glutaminase antibody [EP7212] (ab156876) at 10 µg (purified)

Lane 1 : Human fetal kidney tissue lysate
Lane 2 : HeLa whole cell lysate
Lane 3 : HEK293 whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 73 kDa
Observed band size: 65,73 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM /TBST.

Recognizes endogenous levels of total Glutaminase protein (both the 73 kDa KGA and 65 kDa GAC isoforms).

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Glutaminase with unpurified ab156876 antibody at a dilution of 1/250.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human glioma tissue labelling Glutaminase with purified ab156876 at a dilution of 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

All lanes : Anti-Glutaminase antibody [EP7212] (ab156876) at 1/1000 dilution (unpurified)

Lane 1 : Human fetal brain extract
Lane 2 : Human fetal kidney extract
Lane 3 : HeLa cell lysate
Lane 4 : 293T cell lysate

Lysates/proteins at 10 µg per lane.

Developed using the ECL technique.

Predicted band size: 73 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal human tonsil tissue labelling Glutaminase with unpurified ab156876.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal human brain tissue labelling Glutaminase with unpurified ab156876.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labelling Glutaminase with unpurified ab156876 at a dilution of 1/100.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human pancreas tissue labelling Glutaminase with unpurified ab156876 at a dilution of 1/100.

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

Unpurified ab156876 showing negative staining in Human skeletal muscle.

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

Unpurified ab156876 showing negative staining in Human heart.

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