ab99352 stained HepG2 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 ab99352 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 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.
All lanes : Anti-Anillin antibody (ab99352) at 0.04 µg/mlLane 1 : HeLa whole cell lysate at 50 µgLane 2 : HeLa whole cell lysate at 15 µgLane 3 : HeLa whole cell lysate at 5 µgLane 4 : 293T cell lysate at 50 µgdeveloped using the ECL technique
Detection of Human Anillin by Western Blot and Immunoprecipitation. Samples: Whole cell lysate from HeLa (1 mg for IP, 20% of IP loaded). ab99352 used for WB at 1 µg/ml and used for IP at 3 µg/mg lysate (lane 1). Anillin was also immunoprecipitated by another rabbit anti-Anillin antibody (lane 2) and control IgG (lane 3). Detection Chemiluminescence with exposure times of 3 seconds