Anti-Glutaminase C antibody [EPR19525]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Glutaminase C antibody [EPR19525] (AB202027)

Flow cytometry overlay histogram showing left wild-type HAP1 positive cells and right negative GLS knockout HAP1 stained with ab202027 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab202027) (1x 10⁶ in 100μl at 1 μg/ml (1/603)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C.

Isotype control antibody was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (black line) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

This antibody gave a positive signal in wild-type HAP1 fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Glutaminase C antibody [EPR19525] (AB202027)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Glutaminase C with ab202027 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing mitochondrial staining on HeLa cell line.

The nuclear counter stain is DAPI (blue). COX IV is detected with ab33985 (anti-COX IV(mouse mAb)) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Glutaminase C antibody [EPR19525] (AB202027)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Glutaminase C with ab202027 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Glutaminase C antibody [EPR19525] (AB202027)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Glutaminase Cwith ab202027 at 1/600 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluorr^® 488) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Glutaminase C antibody [EPR19525] (AB202027)

Immunofluorescent analysis of 4% PFA-fixed, 0.1% Triton X-100 permeabilized Hap1 WT and Hap1-GLS KO cells labelling Glutaminase C with ab202027 at 0.2 μg/ml concentration followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 μg/ml dilution (Green). Image showing cytoplasmic staining in Hap1 WT cell line. ab7291 Anti-alpha Tubulin antibody [DM1A] was used to counterstain tubulin at 1/1000 dilution (Magenta). The nuclear counterstain was DAPI (Blue).

• IP

Supplier Data

Immunoprecipitation - Anti-Glutaminase C antibody [EPR19525] (AB202027)

Glutaminase was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab202027 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab202027 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HeLa whole cell lysate, 10 μg (Input).

Lane 2 : ab202027 IP in HeLa whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab202027 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-Glutaminase C antibody [EPR19525] (ab202027)

Observed band size: 65 kDa

false

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Glutaminase C antibody [EPR19525] (AB202027)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling Glutaminase C with ab202027 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on NIH/3T3 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• WB

Supplier Data

Western blot - Anti-Glutaminase C antibody [EPR19525] (AB202027)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Glutaminase C antibody [EPR19525] (ab202027) at 1/1000 dilution

Lane 1:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

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

Lane 3:

K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate at 20 µg

Lane 4:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 5:

Hepa1-6 (Mouse epithelial hepatoma 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 1/100000 dilution

Predicted band size: 65 kDa

Observed band size: 65 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-Glutaminase C antibody [EPR19525] (AB202027)

Blocking/Dilution buffer : 5% NFDM/TBST.

The observed expression profile is consistent with what has been described in UniProtKB (Glutaminase C is highly expressed in heart and pancreas, but is not detected in liver).

All lanes:

Western blot - Anti-Glutaminase C antibody [EPR19525] (ab202027) at 1/1000 dilution

Lane 1:

Human fetal heart lysate at 10 µg

Lane 2:

Human fetal kidney lysate at 10 µg

Lane 3:

Human fetal liver lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 65 kDa

Observed band size: 65 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-Glutaminase C antibody [EPR19525] (AB202027)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure times : Lane 1 and 2 : 3 minutes; Lane 3 : 10 seconds; Lane 4, 5 and 6 : 3 minutes; Lane 7 : 30 seconds; Lane 8, 9 and 10 : 15 seconds.

All lanes:

Western blot - Anti-Glutaminase C antibody [EPR19525] (ab202027) at 1/5000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse heart lysate at 10 µg

Lane 3:

Mouse spleen lysate at 10 µg

Lane 4:

Rat brain lysate at 10 µg

Lane 5:

Rat heart lysate at 10 µg

Lane 6:

Rat spleen lysate at 10 µg

Lane 7:

C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

Lane 8:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 9:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 10:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 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: 65 kDa

Observed band size: 65 kDa

false