IHC-P image of Osteoprotegerrin staining on Rat kidney sections using ab73400 (1:4000). The sections were deparaffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 mins at 21°C. ab73400 was diluted 1:4000 in TBS buffer (containing BSA and Azide) and sections were then incubated with ab73400 for 2 hours at 21°C. The secondary antibody used was Biotin conjugated Goat polyclonal to Rabbit IgG (1:250).

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All lanes : Anti-Osteoprotegerin antibody (ab73400) at 1 µg/ml

Lane 1 : SK N BE (Human neuroblastoma) Whole Cell Lysate
Lane 2 : K562 (Human erythromyeloblastoid leukemia cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size: 46 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?
Additional bands at: 105 kDa, 14 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 8 minutes


Osteoprotegerin contains a number of potential glycosylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.

IHC image of Osteoprotegerin staining in Human Normal Kidney FFPE section, performed on a 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 ab73400, 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.

ICC/IF image of ab73400 stained MCF7 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 (ab73400, 5µg/ml) overnight at +4°C. 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) HeLa, Hek293 and HepG2 cells at 5µg/ml.