SKA3 Antibody / Spindle and kinetochore associated complex subunit 3 / C13orf3 (FY13248)

Immunohistochemical staining of SKA3 using anti-SKA3 antibody. SKA3 was detected in a paraffin-embedded section of human testis tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 ug/ml rabbit anti-SKA3 antibody overnight at 4oC. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using an HRP secondary and DAB substrate.

Western blot analysis of SKA3 using anti-SKA3 antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human 293T whole cell lysates, Lane 2: human Jurkat whole cell lysates, Lane 3: human HEL 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-SKA3 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. Western blot analysis of SKA3 in 293T, Jurkat, and HeLa whole cell lysates using an anti SKA3 antibody. A dominant band at an approximately 50 kDa is detected in all three cell lines, consistent with full length SKA3, which is known to migrate above its predicted 46 kDa mass due to extensive phosphorylation and glycosylation reported in the literature. Additional immunoreactive bands in the low 30 kDa range in all samples and a weaker ~40 kDa band in Jurkat likely represent truncated SKA3 isoforms or proteolytic fragments, or less likely cross reactive proteins.