SPAG16 Antibody / Sperm-associated antigen 16 (FY12458)

Flow Cytometry analysis of SH-SY5Y cells using anti-SPAG16 antibody. Overlay histogram showing SH-SY5Y 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-SPAG16 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 (Red line) was also used as a control.

Western blot analysis of SPAG16 using anti-SPAG16 antibody. Lane 1: human RT4 whole cell lysates, Lane 2: human Hela whole cell lysates, Lane 3: human HepG2 whole cell lysates, Lane 4: human SH-SY5Y whole cell lysates, Lane 5: rat testis tissue lysates, Lane 6: rat C6 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-SPAG16 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. SPAG16 (~71 kDa predicted) was detected at ~65 kDa, consistent with expression of the shorter SPAG16S isoform predominating in somatic and ciliated cells rather than the testis-specific SPAG16L form.