All lanes : Anti-PEX11B antibody (ab74507) at 1/500 dilution
Lane 1 : extracts from COLO cells Lane 2 : extracts from Jurkat
cells Lane 3 : extracts from HUVEC cells Lane 4 : extracts from COS-7 cells Lane 5 : extracts from COLO cells with immunising peptide at 10 µg
Lysates/proteins at 10 µg per lane.
Predicted band size : 28 kDa Observed band size : 28 kDa Additional bands at : 48 kDa,55 kDa,75 kDa. We are unsure as to the identity of these extra bands.
ICC/IF image of ab74507 stained HeLa cells. The cells were 4% formaldehyde fixed (10 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 (ab74507, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit 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.
IHC image of ab74507 staining in human normal colon 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 EDTA (pH9, epitope retrieval solution 2) for 20 mins. The section was then incubated with ab74507, 10µg/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.