Anti-Cathepsin D 抗体 [EPR3056Y] (ab75811)

Anti-Cathepsin D antibody [EPR3056Y] (ab75811) at 1/1000 dilution + MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 15 µg

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

Predicted band size: 44 kDa
Observed band size: 14,44 kDa why is the actual band size different from the predicted?

All lanes : Anti-Cathepsin D antibody [EPR3056Y] (ab75811) at 1/1000 dilution (Unpurified)

Lane 1 : Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 2 : CTSD knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 3 : Hep G2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate

Lysates/proteins at 40 µg per lane.

Performed under reducing conditions.

Predicted band size: 44 kDa
Observed band size: 44 kDa

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.

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.

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).

 

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.