Alexa Fluor® 647 Anti-Glucose Transporter GLUT1 antibody [EPR3915]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Alexa Fluor® 647 Anti-Glucose Transporter GLUT1 antibody [EPR3915] (AB195020)

ab195020 staining Glucose Transporter GLUT1 in HepG2 (human liver hepatocellular carcinoma cell line) cells. The cells were fixed with 4% formaldehyde (10 min), permeabilised in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab195020 at a working dilution of 1 in 50 (shown in red) and ab195887, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor^® 488, shown in green) at 2μg/ml overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

This product gave a positive signal in 100% methanol (5 min) fixed HepG2 cells under the same testing conditions.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Alexa Fluor® 647 Anti-Glucose Transporter GLUT1 antibody [EPR3915] (AB195020)

Overlay histogram showing HepG2 (human liver hepatocellular carcinoma cell line) cells stained with ab195020 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab195020, 1/500 dilution) for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) Alexa Fluor^® 647 used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a solid-state 25mW red diode laser (635 nm) and 675/30 bandpass filter.

This antibody gave a positive signal in HepG2 fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Alexa Fluor® 647 Anti-Glucose Transporter GLUT1 antibody [EPR3915] (AB195020)

Flow cytometry overlay histogram showing wild-type A-549 (green line) and SLC2A1 knockout A-549 stained with ab195020 (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 (ab195020) (1x 10⁶ in 100μl at 0.008 μg/ml (1/62500)) for 30min at 22°C.

Isotype control antibody was Alexa Fluor® 647 Rabbit IgG, monoclonal [EPR25A] - Isotype Control in A-549 WT cells (black line) and A-549-SLC2A1 KO cells (grey line), used at the same concentration and conditions as the primary antibody.

Acquisition of >5000 events were collected using a 40 mW Red laser (638nm) and 660/10 bandpass filter.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Alexa Fluor® 647 Anti-Glucose Transporter GLUT1 antibody [EPR3915] (AB195020)

Immunofluorescent analysis of 4% PFA-fixed 0.1% Triton X-100 permeabilized A549 WT and A549-SLC2A1 KO cells labelling Glucose Transporter GLUT1 with ab195020 at 1 μg/ml concentration (Magenta). Cells were counterstained with ab195887 Anti-Tubulin (Alexa Fluor® 488) at 1 : 250 (2 μg/ml) (Green). The nuclear counterstain was DAPI (Blue).