HRP Anti-Glucose Transporter GLUT1 antibody [EPR3915]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - HRP Anti-Glucose Transporter GLUT1 antibody [EPR3915] (AB195021)

IHC image of Glucose Transporter GLUT1 staining in a section of formalin-fixed paraffin-embedded normal human colon tissue*, performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab195021 at 1/100 dilution, for 15 mins at room temperature. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• WB

Lab

Western blot - HRP Anti-Glucose Transporter GLUT1 antibody [EPR3915] (AB195021)

Western blot : HRP Anti-Glucose Transporter GLUT1 antibody [EPR3915] staining at 1/5000 dilution, shown in black; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab195021 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 before development with Optiblot (ECL reagent ab133456) and imaged with 4 minutes exposure time. Secondary antibodies used were Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - HRP Anti-Glucose Transporter GLUT1 antibody [EPR3915] (ab195021) at 1/5000 dilution

Lane 1:

Wild-type HepG2 cell lysate at 20 µg

Lane 2:

SLC2A1 knockout HepG2 cell lysate at 20 µg

Lane 3:

A549 cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Observed band size: 50-300 kDa

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• WB

Lab

Western blot - HRP Anti-Glucose Transporter GLUT1 antibody [EPR3915] (AB195021)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab195021 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.

All lanes:

Western blot - HRP Anti-Glucose Transporter GLUT1 antibody [EPR3915] (ab195021) at 1/5000 dilution

Lane 1:

Brain (Mouse) Tissue Lysate at 10 µg

Lane 2:

Brain (Human) Tissue Lysate - fetal normal tissue at 10 µg

Predicted band size: 54 kDa

Observed band size: 42 kDa

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Exposure time: 30s