Overlay histogram showing HepG2 cells stained with ab129729 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab129729, 1μg/1x106 cells) for 30 min at 22°C. The secondary antibody used was an anti-mouse Alexa Fluor® 488 (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
IHC image of UGT staining in Human Normal liver formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ 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 ab129729, 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.
Immunoprecipitation image of ab129729. UGT antibody immunocaptured 57 kDa UGT from Human liver homogenate (HLH). Gel stained with Coomassie brilliant blue G.
Lane 1: Ladder
Lane 2: Human liver homogenate lysate
Immunocytochemistry image of ab129729 stained Hela cells. The cells were paraformaldehyde fixed (4%, 20 min) and Triton X-100 permeabilized (0.1%, 15min). The cells were incubated with ab129729 at 5 µg/ml for 2h at room temperature or over night at 4°C. The secondary antibody was (red) AlexaFluor® 594 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. 1% BSA was used as the blocking agent for all blocking steps. DAPI was used to stain the cell nuclei (blue). The target protein locates to the endoplasmic reticulum.