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Western blot - Angiotensin II Type 2 Receptor antibody (ab78747)
All lanes : Anti-Angiotensin II Type 2 Receptor antibody (ab78747) at 1 µg/ml
Lane 1 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 2 :
Lane 3 :
Lane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 41 kDa
Observed band size : 58 kDa (why is the actual band size different from the predicted?)
Exposure time : 5 minutes
Angiotensin II Type 2 Receptor contains a number of potential glycosylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.
Immunocytochemistry/ Immunofluorescence - Angiotensin II Type 2 Receptor antibody (ab78747)
ICC/IF image of ab78747 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 (ab78747, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit 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. This antibody also gave a positive result in 100% methanol fixed (5 min) Hek293, HepG2 and MCF7 cells at 5µg/ml, and in 4% PFA fixed (100 min) HeLa, Hek293, HepG2 and MCF7 cells at 5µg/ml.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Angiotensin II Type 2 Receptor antibody (ab78747)
IHC image of Angiotensin II Type 2 Receptor staining in human normal kidney 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 ab78747, 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.
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