Bone from mouse foot (bone marrow with osteoclasts (positive). IHC-P image was obtained after deparaffinization, antigen retrieval with 0.1 M EDTA (6-8 min, 72 C), drying of slide before staining (10 min, 62 C), and subsequent staining with primary antibody (1/100) in 1% horse serum (1 h, 37 C).

Anti-Cathepsin K antibody [3F9] (ab37259) at 1 µg/ml + Human bone tumor tissue lysate - total protein (ab29359) at 10 µg

Secondary
Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 37 kDa
Observed band size: 37 + 40 kDa why is the actual band size different from the predicted?
Additional bands at: 26 kDa (possible cleavage fragment)


Exposure time: 1 minute


Cathepsin K contains a potential glycosylation site (SwissProt) which may explain its migration at a higher molecular weight than predicted, seen at 40 kDa. The band observed at 25 kDa could potentially be a cleaved form of Cathepsin K due to the presence of both a 15 amino acid signal prptide and a 99 amino acid propeptide. Cathepsin K in its mature form has an expected molecular weight of 25 kDa.

Overlay histogram showing U20S cells stained with ab37259 (red line). The cells were fixed with 80% methanol (5 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. The cells were then incubated with the antibody (ab37259, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x10⁶ cells ) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in U20S cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.