Anti-Cathepsin D 抗体 [EPR3057Y]

• WB

Lab

Western blot - Anti-Cathepsin D antibody [EPR3057Y] (AB75852)

Blocking/Dilution buffer : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Cathepsin D antibody [EPR3057Y] (ab75852) at 1/5000 dilution

All lanes:

Mouse brain tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

Predicted band size: 44 kDa

Observed band size: 28 kDa,43 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cathepsin D antibody [EPR3057Y] (AB75852)

Immunohistochemical analysis of formalin fixed paraffin embedded human liver labelling Cathepsin D with ab75852 at a concentration of 0.01µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab75852 Anti-Cathepsin D antibody [EPR3057Y] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cathepsin D antibody [EPR3057Y] (AB75852)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labeling Cathepsin D with unpurified ab75852 at a dilution of 1/500.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cathepsin D antibody [EPR3057Y] (AB75852)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labeling Cathepsin D with unpurified ab75852 at a dilution of 1/500.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Cathepsin D antibody [EPR3057Y] (AB75852)

Immunocytochemistry/Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma cell line) cells labeling Cathepsin D with purified ab75852 at 1/100.

Cells were fixed with 100% methanol and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1 : primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Cathepsin D antibody [EPR3057Y] (AB75852)

Intracellular Flow Cytometry analysis of HepG2 (Human liver hepatocellular carcinoma cell line)cells labeling Cathepsin D with purified ab75852 at 1/20 dilution (10μg/ml) (red).

Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 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.

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Cathepsin D antibody [EPR3057Y] (AB75852)

IHC image of Cathepsin D staining in a section of frozen normal human liver performed on a Leica BOND^TM system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab75852, 1/1000 dilution, 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. The inset secondary-only control image is taken from an identical assay without primary antibody.

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cathepsin D antibody [EPR3057Y] (AB75852)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labeling Cathepsin D with purified ab75852 at a dilution of 1/100.

Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, an HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500).

Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• IP

Unknown

Immunoprecipitation - Anti-Cathepsin D antibody [EPR3057Y] (AB75852)

ab75852 (purified) at 1/20 immunoprecipitating Cathepsin D in SK-BR-3 (Human mammary gland adenocarcinoma cell line) whole cell lysate.

Lane 1 (input) : SK-BR-3 whole cell lysate (10μg)

Lane 2 (+) : ab75852 + SK-BR-3 whole cell lysate (10μg).

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab75852 in SK-BR-3 whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Blocking/Dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Cathepsin D antibody [EPR3057Y] (ab75852)

Predicted band size: 44 kDa

Observed band size: 28 kDa,43 kDa,46 kDa

false

• WB

Unknown

Western blot - Anti-Cathepsin D antibody [EPR3057Y] (AB75852)

Bands at 28kDa are the Cathepsin D heavy chain.

All lanes:

Western blot - Anti-Cathepsin D antibody [EPR3057Y] (ab75852) at 1/10000 dilution

Lane 1:

MCF7 (Human breast adenocarcinoma cell line) cell lysate at 10 µg

Lane 2:

A431 (Human epidermoid carcinoma cell line) cell lysate at 10 µg

Lane 3:

SK-BR-3 (Human mammary gland adenocarcinoma cell line) cell lysate at 10 µg

Lane 4:

HepG2 (Human liver hepatocellular carcinoma cell line) cell lysate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 44 kDa

Observed band size: 28 kDa,46 kDa

false

• WB

Lab

Western blot - Anti-Cathepsin D antibody [EPR3057Y] (AB75852)

Blocking/Dilution buffer : 5% NFDM /TBST.

This antibody detects 28kDa Cathepsin D heavy chain, 43kDa intermediate form and 46kDa pro-Cathepsin D form. We are unsure of the identity of the band around 75kDa.

All lanes:

Western blot - Anti-Cathepsin D antibody [EPR3057Y] (ab75852) at 1/5000 dilution

Lane 1:

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

Lane 2:

SK-BR-3 (Human mammary gland adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 3:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

Predicted band size: 44 kDa

Observed band size: 28 kDa,43 kDa,46 kDa

false