Anti-Cathepsin D antibody [EPR3056Y]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Cathepsin D antibody [EPR3056Y] (AB75811)

Intracellular Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling Cathepsin D with purified ab75811 at 1/20 dilution (10 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Cathepsin D antibody [EPR3056Y] (AB75811)

Immunocytochemistry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling Cathepsin D with purified ab75811 at 1/50 dilution (1.88 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cathepsin D antibody [EPR3056Y] (AB75811)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid cancer tissue sections labeling Cathepsin D with purified ab75811 at 1/100 dilution (0.94 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 1 (pH 6.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• WB

Lab

Western blot - Anti-Cathepsin D antibody [EPR3056Y] (AB75811)

Lanes 1 - 4 : Merged signal (red and green). Green - ab75811 observed at 44 kDa. Red - loading control, ab7291, observed at 55 kDa.

ab75811 was shown to specifically react with 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% NF Milk. ab75811 and ab7291 (Mouse anti Tubulin loading control) were incubated overnight at 4°C at 1/1000 dilution 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 [EPR3056Y] (ab75811) at 1/1000 dilution

Lane 1:

Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 40 µg

Lane 2:

CTSD knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 40 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:

Hep G2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 40 µg

Lane 4:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 40 µg

Predicted band size: 44 kDa

Observed band size: 44 kDa

false

• WB

Unknown

Western blot - Anti-Cathepsin D antibody [EPR3056Y] (AB75811)

All lanes:

Western blot - Anti-Cathepsin D antibody [EPR3056Y] (ab75811) at 1/1000 dilution

All lanes:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 15 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/20000 dilution

Predicted band size: 44 kDa

Observed band size: 14 kDa,44 kDa

false