Anti-Glucose Transporter GLUT1 antibody [SP168]

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-Glucose Transporter GLUT1 antibody [SP168] (AB150299)

HCT116 WT, SLC2A1 KO were labelled with a green dye. SLC2A1 KOOE cells were labelled with a green and a red dye. SLC2A1 expression was induced with doxycycline in KOOE cells. WT and KO cells were stained with ab150299 and with Alexa-fluor 594 coupled secondary antibody. KOOE cells were stained with : i) ab150299 and with Alexa-fluor 594 coupled secondary antibody; ii) anti-HA-Tag antibody and with Alexa-fluor 488 antibody. Acquisition of the green (HA-Tag in KOOE) and red (antibody staining in WT, KO and KOOE) channels was performed. Representative images of the green and red channel are shown. The antibody used and tested dilution are as follows : ab150299 at 1/50, 1/100 and 1/200.

This image was provided, with thanks, by RESOLUTE (re-solute.eu).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Glucose Transporter GLUT1 antibody [SP168] (AB150299)

Flow Cytometry analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling Glucose Transporter GLUT1 with purified ab150299 at 1/200 dilution (1.24 μg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) / Black. Unlabeled control - Unlabelled cells / blue.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glucose Transporter GLUT1 antibody [SP168] (AB150299)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human lung tissue sections labeling Glucose Transporter GLUT1 with ab150299 at 1/200 dilution (1.24 µg/ml). Heat mediated antigen retrieval was performed Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 10 mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Glucose Transporter GLUT1 antibody [SP168] (AB150299)

Immunocytochemistry/ Immunofluorescence analysis of HepG2 (human hepatocellular carcinoma epithelial cell) cells labeling Glucose Transporter GLUT1 with purified ab150299 at 1/100 (2.5 μg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glucose Transporter GLUT1 antibody [SP168] (AB150299)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human lung carcinoma tissue sections labeling Glucose Transporter GLUT1 with ab150299 at 1/200 dilution (1.24 µg/ml). Heat mediated antigen retrieval was performed Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 10 mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Glucose Transporter GLUT1 antibody [SP168] (AB150299)

Flow cytometry overlay histogram showing wild-type A-549 (green line) and SLC2A1 knockout A-549 stained with ab150299 (magenta line). The cells were fixed with 4% formaldehyde (10 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 (ab150299) (1x 10⁶ in 100μl at 1/12500) 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.

Unlabelled control in A-549 WT cells (black line) and A-549-SLC2A1 KO cells (grey line), at the same conditions as the primary antibody.

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 A-549 fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glucose Transporter GLUT1 antibody [SP168] (AB150299)

Immunohistochemical analysis of formalin fixed, paraffin embedded Human lung carcinoma tissue labelling Glucose Transporter GLUT1 with ab150299 at 1/200 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Glucose Transporter GLUT1 antibody [SP168] (AB150299)

Immunofluorescent analysis of 4% PFA-fixed 0.1% Triton X-100 permeabilized A549 WT and A549-SLC2A1 KO cells labelling Glucose Transporter GLUT1 with ab150299 at 1 μg/ml concentration, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). ab7291 Anti-alpha Tubulin antibody [DM1A] was used to counterstain tubulin at 1/1000 dilution (Magenta). The nuclear counterstain was DAPI (Blue).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glucose Transporter GLUT1 antibody [SP168] (AB150299)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse lung tissue sections labeling Glucose Transporter GLUT1 with ab150299 at 1/200 dilution (1.24 µg/ml). Heat mediated antigen retrieval was performed Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 10 mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• WB

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Western blot - Anti-Glucose Transporter GLUT1 antibody [SP168] (AB150299)

All lanes:

Western blot - Anti-Glucose Transporter GLUT1 antibody [SP168] (ab150299) at 1/200 dilution

All lanes:

HepG2 cell lysate

Predicted band size: 54 kDa

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• WB

Lab

Western blot - Anti-Glucose Transporter GLUT1 antibody [SP168] (AB150299)

Western blot : Anti-Glucose Transporter GLUT1 antibody [SP168] staining at 1/200 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab150299 was shown to bind specifically to Glucose Transporter GLUT1. A band was observed at 50-300 kDa in wild-type HepG2 cell lysates with no signal observed at this size in SLC2A1 knockout cell line ab280797 (knockout cell lysate ab284224). To generate this image, wild-type and SLC2A1 knockout HepG2 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Glucose Transporter GLUT1 antibody [SP168] (ab150299) at 1/200 dilution

Lane 1:

Wild-type HepG2 cell lysate at 20 µg

Lane 2:

SLC2A1 knockout HepG2 cell lysate at 20 µg

Lane 3:

A549 cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Observed band size: 50-300 kDa

false