WB
Western blot analysis of Glucose Transporter GLUT1 on different lysates with Rabbit anti-Glucose Transporter GLUT1 antibodyat 1/50,000 dilution.
Lane 1: HeLa cell lysate (no heat)
Lane 2: HT-29 cell lysate (no heat)
Lane 3: HepG2 cell lysate (no heat)
Lane 4: NIH/3T3 cell lysate (no heat)
Lane 5: L-929 cell lysate (no heat)
Lane 6: Mouse brain tissue lysate (no heat)
Lane 7: Rat brain tissue lysate (no heat)
Lysates/proteins at 20 µg/Lane.
Predicted band size: 54 kDa
Observed band size: 45-60 kDa
Exposure time: Lane 1-7 : 20 seconds
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/50,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1:50,000 dilution was used for 1 hour at room temperature.
IHC
Immunohistochemical analysis of paraffin-embedded human kidney tissue using anti-Glucose Transporter GLUT1 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (1/200) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ICC/IF
Immunocytochemistry analysis of NIH/3T3 cells labeling Glucose Transporter GLUT1 with Rabbit anti-Glucose Transporter GLUT1 antibody at 1/500 dilution.
Cells were fixed in 100% precooled methanol for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Glucose Transporter GLUT1 antibody at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594) was used as the secondary antibody at 1/1,000 dilution.
FC
Flow cytometric analysis of HepG2 cells labeling Glucose Transporter GLUT1.
Cells were fixed and permeabilized. Then stained with the primary antibody (1ug/ml) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).