Anti-Glucose Transporter GLUT1 抗体 [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).

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.

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.

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.