BNIP3L Antibody / NIX / BCL2/adenovirus E1B 19 kDa-interacting protein 3-like (FY13212)

Immunofluorescent staining of BNIP3L using anti-BNIP3L antibody (green). BNIP3L was detected in an immunocytochemical section of U2OS 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-BNIP3L 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. The section was counterstained with DAPI nuclear stain (blue). Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of BNIP3L using anti-BNIP3L antibody. Lane 1: human 293T whole cell lysates, Lane 2: human Jurkat whole cell lysates, Lane 3: human K562 whole cell lysates, Lane 4: rat kidney tissue lysates, Lane 5: mouse kidney tissue 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-BNIP3L 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. Western blot detection of BNIP3L (NIX) shows a single band at ~35 kDa across cell and tissue lysates. Despite a calculated mass of ~24-26 kDa, BNIP3L commonly migrates at ~32-36 kDa due to its transmembrane domain and phosphorylation-dependent mobility.

Flow Cytometry analysis of human JK cells using anti-BNIP3L antibody. Overlay histogram showing JK 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-BNIP3L 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.