All lanes : Anti-Topoisomerase III alpha antibody (ab108493) at 1 µg/mlLane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell LysateLane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell LysateLane 3 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with Immunizing peptide at 1 µg/mlLane 4 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate with Immunizing peptide at 1 µg/mlLysates/proteins at 10 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
ab108493 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab108493 at 1µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.