Anti-Cathepsin B 抗体 (ab92955)

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: Cathepsin B knockout HAP1 whole cell lysate (20 µg)
Lane 3: A549 whole cell lysate (20 µg)
Lane 4: HL60 whole cell lysate (Negative Control) (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab92955 observed at 24-27-44 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab92955 was shown to recognize Cathepsin B in wild-type HAP1 cells as signal was lost at the expected MW in Cathepsin B knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and Cathepsin B knockout samples were subjected to SDS-PAGE. ab92955 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Pro-cathepsin B runs at approximately 44 kDa on SDS-PAGE. When it is proteolytically processed and glycosylated it forms a mature two-chain protein containing a heavy chain (running at 27 and 24 kDa) and a light chain (5 kDa)

ICC/IF image of ab92955 stained HepG2 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 ab92955 at 1µg/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.