SUN1 Antibody / Sad1/UNC-84 domain-containing protein 1 (FY12319)

Flow Cytometry analysis of Jurkat cells using anti-SUN1 antibody. Overlay histogram showing human Jurkat 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-SUN1 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.

Western blot analysis of SUN1 using anti-SUN1 antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human whole cell lysates, Lane 2: human Jurkat whole cell lysates, Lane 3: human K562 whole cell lysates, Lane 4: human Hela 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-SUN1 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. The expected molecular weight of SUN1 is ~87 kDa with multiple smaller and one larger isoform. A ~100 kDa band for SUN1 is consistent with published literature. It most likely reflects a post-translationally modified or longer isoform of SUN1 rather than a detection error. Multiple studies report similar upward shifts and multiple SUN1 bands in mammalian cell lysates.