Anti-Cathepsin D antibody (ab72915)
Key features and details
- Rabbit polyclonal to Cathepsin D
- Suitable for: WB, ICC/IF, IHC-P
- Knockout validated
- Reacts with: Human
- Isotype: IgG
Overview
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Product name
Anti-Cathepsin D antibody
See all Cathepsin D primary antibodies -
Description
Rabbit polyclonal to Cathepsin D -
Host species
Rabbit -
Tested Applications & Species
See all applications and species dataApplication Species IHC-P HumanWB Human
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Immunogen
Synthetic peptide corresponding to Human Cathepsin D aa 200-300 conjugated to keyhole limpet haemocyanin.
(Peptide available asab90605) -
Positive control
- WB: MCF7, A431 and HepG2 whole cell lysates. ICC/IF: methanol fixed HepG2 cells. IHC-P: human adrenal gland tissue.
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General notes
Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.
Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.
We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.
In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.
We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.
Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.
Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.40
Preservative: 0.02% Sodium azide
Constituent: PBS
Batches of this product that have a concentration
Concentration information loading...Purity
Immunogen affinity purifiedClonality
PolyclonalIsotype
IgGResearch areas
Associated products
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Compatible Secondaries
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Isotype control
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Recombinant Protein
Applications
The Abpromise guarantee
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.
GuaranteedTested applications are guaranteed to work and covered by our Abpromise guarantee.
PredictedPredicted to work for this combination of applications and species but not guaranteed.
IncompatibleDoes not work for this combination of applications and species.
Application Species IHC-P HumanWB HumanAll applications DogPigOrangutanApplication Abreviews Notes WB (1) 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.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
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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
modificationsN- 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
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Database links
- Entrez Gene: 1509 Human
- Omim: 116840 Human
- SwissProt: P07339 Human
- Unigene: 654447 Human
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Alternative names
- CatD antibody
- CATD_HUMAN antibody
- Cathepsin D antibody
see all
Images
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Western blot - Anti-Cathepsin D antibody (ab72915)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 kDaLanes 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.
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Western blot - Anti-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
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. -
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.
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Immunocytochemistry/ Immunofluorescence - Anti-Cathepsin D antibody (ab72915)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.
Protocols
Datasheets and documents
References (5)
ab72915 has been referenced in 5 publications.
- Xu Y et al. YWHA/14-3-3 proteins recognize phosphorylated TFEB by a noncanonical mode for controlling TFEB cytoplasmic localization. Autophagy 15:1017-1030 (2019). PubMed: 30653408
- 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). 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 . PubMed: 27994678
- Jansson PJ et al. Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes multidrug resistance by a novel mechanism involving the hijacking of lysosomal P-glycoprotein (Pgp). J Biol Chem 290:9588-603 (2015). PubMed: 25720491
- Gutierrez E et al. The anticancer agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes prosurvival autophagy by two mechanisms: persistent induction of autophagosome synthesis and impairment of lysosomal integrity. J Biol Chem 289:33568-89 (2014). PubMed: 25301941
Images
-
Western blot - Anti-Cathepsin D antibody (ab72915)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 kDaLanes 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.
-
Western blot - Anti-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
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. -
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.
-
Immunocytochemistry/ Immunofluorescence - Anti-Cathepsin D antibody (ab72915)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.
