ICC/IF image of ab72028 stained DU145 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 ab72028 at 5µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
Western blot - PPP2R5A antibody (ab72028)
All lanes : Anti-PPP2R5A antibody (ab72028) at 0.04 µg/ml
Lane 1 : Whole cell lysate from Hela cells at 50 µg Lane 2 : Whole cell lysate from Hela cells at 15 µg Lane 3 : Whole cell lysate from Hela cells at 5 µg Lane 4 : Whole cell lysate from 293T cells at 50 µg Lane 5 : Whole cell lysate from NIH 3T3 cells at 50 µg
Predicted band size : 56 kDa Observed band size : 56 kDa Additional bands at : 250 kDa. We are unsure as to the identity of these extra bands.
Immunoprecipitation - PPP2R5A antibody (ab72028)
Immunoprecipitation/ Western Blot of PPP2R5A.
Lane 1: ab72028 at 3µg/mg whole cell lysate.
Lane 2: Control IgG.
Whole cell lysate from Hela cells at 1mg for IP, 20% of IP loaded.
For Western blot detection ab72028 was used at 0.1 µg/ml.
Chemiluminescence with an exposure time of 30 seconds.
IHC image of ab72028 staining in human normal breast formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab72028, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.