Overview

  • Product name

  • Description

    Rabbit polyclonal to Cathepsin D
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, ICC/IF, IHC-Pmore details
  • Species reactivity

    Reacts with: Human
    Predicted to work with: Dog, Pig, Orangutan
  • Immunogen

    Synthetic peptide corresponding to Human Cathepsin D aa 200-300 conjugated to keyhole limpet haemocyanin.
    (Peptide available as ab90605)

  • Positive control

    • WB: MCF7, A431 and HepG2 whole cell lysates. ICC/IF: methanol fixed HepG2 cells. IHC-P: human adrenal gland tissue.

Properties

Applications

Our Abpromise guarantee covers the use of ab72915 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
WB Use a concentration of 1 µg/ml. Detects a band of approximately 45 kDa (predicted molecular weight: 45 kDa).
ICC/IF Use a concentration of 5 µg/ml.
IHC-P Use a concentration of 0.1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

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 (ab72915) at 1 µg/ml

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

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 45 kDa



    Lanes 1 - 4: Merged signal (red and green). Green - ab72915 observed at 28, 43, 46 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab72915 was shown to specifically react with CTSD 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% Milk. Ab72915 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml 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-Cathepsin D antibody (ab72915) at 1 µg/ml

    Lane 1 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
    Lane 2 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 3 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Developed using the ECL technique.

    Predicted band size: 45 kDa
    Observed band size: 29,45,48 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 150 kDa, 90 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 4 minutes


    Cathepsin D has a predicted molecular weight of 45 kDa. The sequence contains a signal sequence and propeptide of 18 and 45 amino acids, respectively. This protein is further cleaved to produce a heavy and light chain with molecular weights of 27 kDa and 11 kDa, respectively (SwissProt). We hypothesize that the observed bands at 29 kDa represent the Cathepsin heavy chain, and the bands at 45 and 48 kDa represent the protein with and without the presence of the signal peptide.
  • IHC image of Cathepsin D staining in human adrenal gland formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab72915, 0.1µg/ml, 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.

    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.

  • ICC/IF image of ab72915 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Triton for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab72915 at 5µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150120 (pseudo-colored red) and ab150081 (colored green) used at 1 µg/ml for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

References

This product has been referenced in:

  • Al-Akra L  et al. Tumor stressors induce two mechanisms of intracellular P-glycoprotein-mediated resistance that are overcome by lysosomal-targeted thiosemicarbazones. J Biol Chem 293:3562-3587 (2018). Read more (PubMed: 29305422) »
  • Wymant JM  et al. The Role of BCA2 in the Endocytic Trafficking of EGFR and Significance as a Prognostic Biomarker in Cancer. J Cancer 7:2388-2407 (2016). IF . Read more (PubMed: 27994678) »
See all 4 Publications for this product

Customer reviews and Q&As

Application
Western blot
Sample
Cat Cell lysate - whole cell (CAT-MT and K12- Feline mammary carcinoma, SCCF1)
Gel Running Conditions
Reduced Denaturing (4-20%)
Loading amount
50 µg
Specification
CAT-MT and K12- Feline mammary carcinoma, SCCF1
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%

Holly Pondenis

Verified customer

Submitted Aug 26 2015

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