UBE2T Antibody / Ubiquitin-conjugating enzyme E2 T / HSPC150 (FY12094)

IF analysis of UBE2T using anti-UBE2T antibody (green). UBE2T was detected in an immunocytochemical section of MCF-7 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 1:100 rabbit anti-UBE2T antibody overnight at 4oC. DyLight 488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37oC. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of HSPC150/UBE2T using anti-UBE2T antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human Hela whole cell lysates, Lane 2: human Skov3 whole cell lysates, Lane 3: human MCF-7 whole cell lysates, Lane 4: human K562 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-UBE2T antibody at 1:1000 overnight at 4oC, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal was developed using an ECL Plus Western Blotting Substrate. A specific band was detected for UBE2T at approximately 23 kDa. The expected band size for UBE2T is at 23 kDa.

Western blot analysis of UBE2T using anti-UBE2T antibody. Electrophoresis was performed on a 12% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human Hela whole cell lysates, Lane 2: human SK-OV-3 whole cell lysates, Lane 3: human MCF-7 whole cell lysates, Lane 4: human K562 whole cell lysates, Lane 5: rat testis tissue lysates, Lane 6: rat RH35 whole cell lysates, Lane 7: mouse testis tissue lysates, Lane 8: mouse RAW264.7 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-UBE2T antibody at 0.5 ug/ml overnight at 4oC, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal was developed using an ECL Plus Western Blotting Substrate. A specific band was detected for UBE2T at approximately 23 kDa. The expected band size for UBE2T is at 23 kDa.

Flow Cytometry analysis of MCF-7 cells using anti-UBE2T antibody. Overlay histogram showing MCF-7 cells stained with (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-UBE2T antibody (1 ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10 ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1 ug/million cells) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.