Recombinant Anti-Glucose Transporter GLUT1 antibody [EPR3915] (ab115730)

Western blot: Anti-Glucose Transporter GLUT1 antibody [EPR3915] staining at 1/100000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab115730 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.

Lanes 1 - 2: Merged signal (red and green). Green - ab196357 observed at 54 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab196357 was shown to recognize in wild-type A549 cells as signal was lost at the expected MW in SLC2A1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and SLC2A1 knockout samples were subjected to SDS-PAGE. Ab196357 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

GLUT1 and GLUT3 are downregulated in KSHV-infected cells in human KS tumors

Representative illustration of dual immunofluorescence detection of LANA and GLUT1 or in a normal human skin section and a Karposi Sarcoma (KS) tumor section. Tissues were fixed with paraformaldehyhde and paraffin-embedded.

Immunohistochemical expression of Glut1 in normal tongue epithelium and tongue cancer. Expression was greatest in lymphocytes (arrows in left upper and lower panels). In the normal oral epithelium, Glut1 was weakly expressed in the basal and spinous cells (left upper panel). In OSCC, Glut1 was upregulated, showing a level of expression comparable with lymphocytes (left and right lower panels). Scale bar, 100 μm.

Note: Glut1 = SLC2A (alternative names for the same target).

Exposure time

Lane 1 to 2: 10 seconds
Lane 3 to 4: 30 seconds

We recommend not to boil the samples after lysis to get desired WB bands.

ab115730 staining SLC2A1 in wild-type A549 cells, with negative expression in SLC2A1 knockout A549 cells. The cells were fixed with 100% methanol (5 min), permeabilised with 0.1% Triton x-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab115730 at 1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 647), pre-adsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

Immunofluorescence staining of HepG2 cells with purified ab115730 at a working dilution of 1/100, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab115730 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

Alexa Fluor^® 488 (ab195359) and Alexa Fluor^® 647 (ab195020) conjugated versions are available for this clone.

Overlay histogram showing Jurkat cells fixed in 4% PFA and stained with purified ab115730 at a dilution of 1/40 (red line). The secondary antibody used was Alexa Fluorr^® 488 goat anti-rabbit at a dilution of 1/500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line). 

Alexa Fluorr^®488 (ab195359) and Alexa Fluorr^®647 (ab195020) conjugated versions are available for this clone. 

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Overlay histogram showing HeLa cells stained with unpurified ab115730 (red line). The cells were fixed with 80% methanol (5 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 (ab115730, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions. 

Unpurified ab115730 at 1/250 dilution staining Glucose Transporter GLUT1 in Paraffin-embedded human cervical carcinoma tissue by Immunohistochemistry.

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

Unpurified ab115730 at 1/250 dilution staining Glucose Transporter GLUT1 in Paraffin-embedded human colonic adenocarcinoma tissue by Immunohistochemistry.

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

Unpurified ab115730 showing positive staining in human normal liver tissue.

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

Unpurified ab115730 showing positive staining in human normal breast tissue.

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

Unpurified ab115730 showing positive staining in human normal colon tissue.

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

Unpurified ab115730 showing positive staining in human kidney carcinoma tissue.

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

Unpurified ab115730 showing negative staining in human skeletal muscle tissue.

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

Unpurified ab115730 showing positive staining in human urinary bladder transitional carcinoma tissue.

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

Unpurified ab115730 showing negative staining in human normal heart tissue.

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