Anti-Cathepsin B antibody

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Cathepsin B antibody (AB92955)

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.

• WB

Lab

Western blot - Anti-Cathepsin B antibody (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)

All lanes:

Western blot - Anti-Cathepsin B antibody (ab92955)

Predicted band size: 38 kDa

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• WB

Unknown

Western blot - Anti-Cathepsin B antibody (AB92955)

We hypothesize that the 25 and 30 kDa bands represent the heavy chain and mature form of Cathepsin B, respectively. Abcam welcomes customer feedback.

All lanes:

Western blot - Anti-Cathepsin B antibody (ab92955) at 1 µg/mL

Lane 1:

Human liver tissue lysate - total protein (<a href='/en-us/products/unavailable/human-liver-tissue-lysate-total-protein-ab29889'>ab29889</a>) at 10 µg

Lane 2:

Human kidney tissue lysate - total protein (<a href='/en-us/products/unavailable/human-kidney-tissue-lysate-total-protein-ab30203'>ab30203</a>) at 10 µg

Lane 3:

Human brain tissue lysate - total protein (<a href='/en-us/products/unavailable/human-brain-tissue-lysate-total-protein-ab29466'>ab29466</a>) at 10 µg

Lane 4:

Human placenta tissue lysate - total protein (<a href='/en-us/products/unavailable/human-placenta-tissue-lysate-total-protein-ab29745'>ab29745</a>) at 10 µg

Lane 5:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 6:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 38 kDa

Observed band size: 25 kDa,30 kDa

true

Exposure time: 30s