KEAP1 Antibody / Kelch-like ECH-associated protein 1 (FY12106)

IF analysis of KEAP1 using anti-KEAP1 antibody (green). KEAP1 was detected in an immunocytochemical section of 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 5 ug/ml rabbit anti-KEAP1 antibody overnight at 4oC. DyLight 488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37oC. The section was counterstained with DAPI (blue). Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of KEAP1 using anti-KEAP1 antibody. Lane 1: human Hela whole cell lysates, Lane 2: human Jurkat whole cell lysates, Lane 3: human 293T whole cell lysates, Lane 4: human HepG2 whole cell lysates, Lane 5: rat C6 whole cell lysates, Lane 6: mouse Neuro-2a 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-KEAP1 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 enhanced chemiluminescent. A specific band was detected for KEAP1 at approximately 66-72 kDa. The expected band size for KEAP1 is at 70 kDa.

Flow Cytometry analysis of U251 cells using anti-KEAP1 antibody. Overlay histogram showing U251 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-KEAP1 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.