All lanes : Anti-Glucose Transporter GLUT1 antibody (ab14683) at 1/2500 dilution

Lane 1 : Human placenta tissue lysate - total protein (ab29745)
Lane 2 : SW480 (Human colon adenocarcinoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 55 kDa
Observed band size: 55 kDa



This antibody labels Glut 1 protein as a smear due to large amount of glycosylation on Glut 1 transporter. We observed that boiling and freezing the Glut 1 samples resulted in aggregated form of the protein and gives a smear form high 100kDa range to 45kDa with significant reduction in Glut 1 (44-47kDa) band. We suggest that samples should not be heated or frozen. Following extraction in lysis buffer, the proteins are then dissolved in SDS-PAGE sample buffer 2X and reduced in presence of 2.5-5% BME. All samples at this time will be heated 70-80°C for 3 minutes and aliquot and stored frozen till it is time for analysis. Just before analyses the samples much be thawed at room temp or at 37°C to dissolve any precipitated SDS in the sample. The remaining sample must not be frozen again, this freeze and next thaw cycle will make aggregate in some samples.

Human normal placenta. Staining is observed at the cell surface.

Left panel: Primary antibody at 2 ug/ml.

Right panel: Isotype control.

Sections were stained using an automated system DAKO Autostainer Plus , at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers citrate pH 6.1 in a DAKO PT Link. Slides were peroxidase blocked in 3% H₂O₂ in methanol for 10 minutess. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako envision flex amplification kit for rabbit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with haematoxylin and coverslipped under DePeX.

Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

ICC/IF image of ab14683 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells. The cells were 4% formaldehyde fixed (10 minutes) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab14683, 5 µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1 hour. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.

Immunohistochemical analysis of mammary glands taken from wild-type or Erbb4^-/- lactating mice, staining Glucose Transporter GLUT1 with ab14683.