All lanes : Anti-proCathepsin D antibody [EPR3054] (ab134169) at 1/2000 dilution

Lane 1 : Wild-type A431 whole cell lysate
Lane 2 : Cathepsin D knockout A431 whole cell lysate
Lane 3 : HepG2 whole cell lysate
Lane 4 : MCF7 whole cell lysate

Lysates/proteins at 40 µg per lane.

Predicted band size: 44 kDa
Observed band size: 46 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab134169 observed at 46 kDa. Red - loading control, ab7291 (mouse anti-tubulin), observed at 50 kDa.

ab134169 was shown to recognize in wild-type A431 cells as signal was lost at the expected MW in CTSD knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CTSD knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF Milk. Ab134169 and ab7291 (Mouse anti-tubulin loading control) were incubated overnight at 4°C at 1/2000 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 : Anti-proCathepsin D antibody [EPR3054] (ab134169) at 1/2000 dilution (purified)

Lane 1 : MCF-7 cell lysate
Lane 2 : A431 cell lysate
Lane 3 : SKBR-3 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

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

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Flow Cytometry analysis of A431 (human epidermoid carcinoma) cells labeling proCathepsin D with purified ab134169 at 1/100 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488)(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

Immunohistochemical staining of paraffin embedded human breast carcinoma with purified ab134169 at a working dilution of 1 in 50. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunofluorescence staining of MCF7 cells with purified ab134169 at a working dilution of 1 in 50, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti rabbit (ab150077), used at a dilution of 1 in 500. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative control is shown in bottom right hand panel - for the negative control, purified ab134169 was used at a dilution of 1/200 followed by an Alexa Fluor^® 594 goat anti-mouse antibody at a dilution of 1/500.

All lanes : Anti-proCathepsin D antibody [EPR3054] (ab134169) at 1/2000 dilution (unpurified)

Lane 1 : MCF7 cell lysate
Lane 2 : A431 cell lysate
Lane 3 : SKBR3 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Standard HRP labelled goat anti-rabbit at 1/2000 dilution

Developed using the ECL technique.

Predicted band size: 44 kDa

Immunohistochemical analysis of paraffin-embedded Human breast ductal infiltrating carcinoma tissue, staining proCathepsin D using unpurified ab134169 at a 1/250 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Equilibrium disassociation constant (K_D)
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