Anti-Cathepsin D antibody

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cathepsin D antibody (AB72915)

IHC image of Cathepsin D staining in human adrenal gland formalin fixed paraffin embedded tissue section, performed on a Leica Bond 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 ab72915, 0.1μg/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.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Cathepsin D antibody (AB72915)

ICC/IF image of ab72915 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-Triton for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab72915 at 5μg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150120 (pseudo-colored red) and ab150081 (colored green) used at 1 μg/ml for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 μM for 1hour at room temperature.

• WB

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Western blot - Anti-Cathepsin D antibody (AB72915)

Cathepsin D has a predicted molecular weight of 45 kDa. The sequence contains a signal sequence and propeptide of 18 and 45 amino acids, respectively. This protein is further cleaved to produce a heavy and light chain with molecular weights of 27 kDa and 11 kDa, respectively (SwissProt). We hypothesize that the observed bands at 29 kDa represent the Cathepsin heavy chain, and the bands at 45 and 48 kDa represent the protein with and without the presence of the signal peptide.

All lanes:

Western blot - Anti-Cathepsin D antibody (ab72915) at 1 µg/mL

Lane 1:

MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 3:

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

Secondary

All lanes:

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

Predicted band size: 44 kDa

Observed band size: 150 kDa,29 kDa,45 kDa,48 kDa,90 kDa

true

Exposure time: 4min

• WB

Lab

Western blot - Anti-Cathepsin D antibody (AB72915)

Lanes 1 - 4 : Merged signal (red and green). Green - ab72915 observed at 28, 43, 46 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab72915 was shown to specifically react with CTSD in wild-type A431 cells as signal was lost in CTSD knockout cells. Wild-type and CTSD knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. ab72915 and ab8245 (Mouse anti-GAPDH 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.

All lanes:

Western blot - Anti-Cathepsin D antibody (ab72915) at 1 µg/mL

Lane 1:

Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

CTSD knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human CTSD knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-ctsd-knockout-a-431-cell-line-ab261891'>ab261891</a>)

Lane 3:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 4:

Hep G2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Predicted band size: 44 kDa

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