The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 45 kDa (predicted molecular weight: 45 kDa).
Use a concentration of 5 µg/ml.
Use a concentration of 0.1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
FunctionAcid protease active in intracellular protein breakdown. Involved in the pathogenesis of several diseases such as breast cancer and possibly Alzheimer disease.
Tissue specificityExpressed in the aorta extrcellular space (at protein level).
Involvement in diseaseCeroid lipofuscinosis, neuronal, 10
Sequence similaritiesBelongs to the peptidase A1 family. Contains 1 peptidase A1 domain.
Post-translational modificationsN- and O-glycosylated.
Cellular localizationLysosome. 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).
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Anti-Cathepsin D antibody images
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cathepsin D antibody (ab72915)
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.
Western blot - Cathepsin D antibody (ab72915)
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 Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution Developed using the ECL technique
Exposure time : 4 minutesCathepsin 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.
Immunocytochemistry/ Immunofluorescence - Anti-Cathepsin D antibody (ab72915)
ICC/IF image of ab72915 stained MCF-7 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-Tween 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 overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
References for Anti-Cathepsin D antibody (ab72915)
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