Overview

  • Product name
    Anti-Cathepsin D antibody [EPR3057Y]
    See all Cathepsin D primary antibodies
  • Description
    Rabbit monoclonal [EPR3057Y] to Cathepsin D
  • Host species
    Rabbit
  • Tested applications
    Suitable for: Flow Cyt, ICC/IF, WB, IP, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide within Human Cathepsin D aa 350 to the C-terminus (C terminal). The exact sequence is proprietary.

  • Positive control
    • WB: MCF7, A431, SK-BR-3 and HepG2 whole cell lysate (ab7900) and mouse brain tissue lysate. IHC-P: Human breast carcinoma and liver tissues. ICC/IF: MCF7 cells. IP: SK-BR-3 cell lysate. Flow Cyt: HepG2 cells.
  • General notes

    Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information. 

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab75852 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Flow Cyt Use at an assay dependent concentration.
ICC/IF 1/100 - 1/1000.
WB 1/2000 - 1/10000. Predicted molecular weight: 46 kDa.
IP 1/10 - 1/20.
IHC-P 1/100 - 1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

Target

  • Function
    Acid protease active in intracellular protein breakdown. Involved in the pathogenesis of several diseases such as breast cancer and possibly Alzheimer disease.
  • Tissue specificity
    Expressed in the aorta extrcellular space (at protein level).
  • Involvement in disease
    Ceroid lipofuscinosis, neuronal, 10
  • Sequence similarities
    Belongs to the peptidase A1 family.
    Contains 1 peptidase A1 domain.
  • Post-translational
    modifications
    N- and O-glycosylated.
  • Cellular localization
    Lysosome. Melanosome. Secreted, extracellular space. Identified by mass spectrometry in melanosome fractions from stage I to stage IV. In aortic samples, detected as an extracellular protein loosely bound to the matrix (PubMed:20551380).
  • Information by UniProt
  • Database links
  • Alternative names
    • CatD antibody
    • CATD_HUMAN antibody
    • Cathepsin D antibody
    • Cathepsin D heavy chain antibody
    • CD antibody
    • Ceroid lipofuscinosis neuronal 10 antibody
    • CLN10 antibody
    • CPSD antibody
    • ctsd antibody
    • Epididymis secretory sperm binding protein Li 130P antibody
    • HEL S 130P antibody
    • Lysosomal aspartyl peptidase antibody
    • Lysosomal aspartyl protease antibody
    • MGC2311 antibody
    see all

Images

  • All lanes : Anti-Cathepsin D antibody [EPR3057Y] (ab75852) at 1/5000 dilution (purified)

    Lane 1 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
    Lane 2 : SK-BR-3 (Human mammary gland adenocarcinoma cell line) whole cell lysate
    Lane 3 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

    Predicted band size: 46 kDa
    Observed band size: 28,43,46 kDa
    why is the actual band size different from the predicted?



    Blocking/Dilution buffer: 5% NFDM /TBST.

     

  • 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).

  • 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.

  • Anti-Cathepsin D antibody [EPR3057Y] (ab75852) at 1/5000 dilution (purified) + Mouse brain tissue lysate at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

    Predicted band size: 46 kDa
    Observed band size: 28,43 kDa why is the actual band size different from the predicted?



    Blocking/Dilution buffer: 5% NFDM /TBST.

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

    Cells were fixed with 4% paraformaldehyde and permeabilized 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.

  • 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, ab131366 VeriBlot for IP (HRP) was used as the secondary antibody (1/10,000).

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

  • All lanes : Anti-Cathepsin D antibody [EPR3057Y] (ab75852) at 1/10000 dilution (unpurified)

    Lane 1 : MCF7 (Human breast adenocarcinoma cell line) cell lysate
    Lane 2 : A431 (Human epidermoid carcinoma cell line) cell lysate
    Lane 3 : SK-BR-3 (Human mammary gland adenocarcinoma cell line) cell lysate
    Lane 4 : HepG2 (Human liver hepatocellular carcinoma cell line) cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

    Predicted band size: 46 kDa
    Observed band size: 46 kDa
    Additional bands at: 28 kDa (possible cleavage fragment)



    Bands at 28kDa are the Cathepsin D heavy chain.
  • 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.

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

References

This product has been referenced in:
  • Milenkovic A  et al. BEST1 protein stability and degradation pathways differ between autosomal dominant Best disease and autosomal recessive bestrophinopathy accounting for the distinct retinal phenotypes. Hum Mol Genet 27:1630-1641 (2018). Read more (PubMed: 29668979) »
  • Härtlova A  et al. LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages. EMBO J 37:N/A (2018). Read more (PubMed: 29789389) »
See all 20 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Application
Western blot
Sample
Dog Cell lysate - whole cell (MDCK2 cells)
Gel Running Conditions
Reduced Denaturing (10%)
Loading amount
20 µg
Treatment
50uM chloroquine for 5h
Specification
MDCK2 cells
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 23°C

Herr Dr. Vladimir Milenkovic

Verified customer

Submitted Oct 24 2018

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Human Tissue sections (adult skin)
Permeabilization
No
Specification
adult skin
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 22°C
Fixative
unfixed

Abcam user community

Verified customer

Submitted Jul 02 2018

Application
Western blot
Sample
Human Cell lysate - whole cell (Primary keratinocytes)
Gel Running Conditions
Reduced Denaturing (any kD BioRad)
Loading amount
50 µg
Specification
Primary keratinocytes
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Mr. Julien Coutier

Verified customer

Submitted Jul 02 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (blood monocyte derived macrophage)
Loading amount
100 µg
Specification
blood monocyte derived macrophage
Gel Running Conditions
Reduced Denaturing (10)
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Jan 18 2013

Answer

Vielen Dank für dieses Update.

Es freut mich zu hören, dass Sie versuchen werden das Ergebnis mit unseren Vorschlägen zu verbessern. Ich weiß, dass es viel Zeit und Organisation im Labor erfordert und schätze Ihren Einsatz.

Bitte melden Sie sich, sollten Sie keine Verbesserung des Ergebnisses erhalten und ich werde für Sie ein Ersatzprodukt oder eine Gutschrift organisieren.

Falls ich vor Weihnachten nicht mehr von Ihnen höre wünsche ich Ihnen ein frohes Fest und kommen Sie gut ins neue Jahr.

Read More

Answer

Vielen Dank für Ihren Anruf und dafür, dass Sie sich die Zeit genommen haben, unseren Fragebogen auszufüllen.

Es tut mir leid, dass Sie Probleme mit unserem Antikörper hatten.

Das verwendete Protokoll sieht gut aus und es gibt leider nicht viel was ich noch vorschlagen kann. In dem Versuch Ihr Problem zu lösen, möchte ich die folgenden Anregungen zu Veränderungen des Experimentes machen.

1. Wenn es möglich ist wäre es hilfreich den Antigen retrival mit der HIER Methode für Ihre Positivkontrolle zu verwenden. Dies würde weitere Rückschlüsse auf die Qualität des Antikörpers ermöglichen und zeigen ob die verwendete Probe den Nachweis von Cathepsin D erschwert.

2. Ich habe keine nähere Information zur Beschaffenheit der Proben gefunden. Wenn es sich dabei um recht dicke Gewebeschnitte handelt könnte ein eingeführter Permeabilisierungsschritt hilfreich sein. Die Zugänglichkeit des Gewebes für den Antikörper könnte dadurch erleichtert werden.

3. Die empfohlene Konzentration für diesen Antikörper ist eine Verdünnung von 1:75 bis 1:100. Je nach erwarteter Expressionsstärke könnte die Erhöhung der Antikörperkonzentration hilfreich sein.

Ich möchte Ihnen noch einmal versichern, dass dieser Antikörper für die IHC-P Anwendung in Ratte für sechs Monate garantiert ist. Wenn Sie weiterhin Probleme haben zögern Sie daher nicht sich wieder bei mir zu melden und ich werde gerne eine Kompensation dieses Antikörpers für Sie arrangieren. Als Kompensation können Sie dasselbe Produkt (anderes Lot wenn vorhanden), ein alternatives Produkt oder eine Gutschrift erhalten.

Zusaetzlich habe ich mich mit dem Labor in Verbindung gesetzt um zusätzliche Informationen den ab75852 und dessen Reaktivität mit Ratte betreffend zu erhalten. Sobald ich eine Rückmeldung erhalte werde ich diese Information an Sie weiter leiten.


Bitte lassen Sie mich wissen, ob ich Ihnen helfen konnte und zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (liver)
Specification
liver
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate pH6.0
Permeabilization
No
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 37°C

Abcam user community

Verified customer

Submitted Jun 11 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (MCF7 cells)
Loading amount
15 µg
Specification
MCF7 cells
Gel Running Conditions
Reduced Denaturing (4-12%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted May 21 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (NIH 3T3 cells)
Loading amount
15 µg
Specification
NIH 3T3 cells
Gel Running Conditions
Reduced Denaturing (4-12%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted May 21 2009

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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