Key features and details
- Assay type: Semi-quantitative
- Detection method: Fluorescent
- Platform: Microplate reader
- Assay time: 2 hr
- Sample type: Cell Lysate, Tissue Extracts
Product nameCathepsin D Activity Assay Kit (Fluorometric)
See all Cathepsin D kits
Sample typeTissue Extracts, Cell Lysate
Assay time2h 00m
Species reactivityReacts with: Mammals, Other species
Cathepsin D Activity Assay Kit ab65302 is a fluorescence-based assay that utilizes the preferred cathepsin-D substrate sequence GKPILFFRLK(Dnp)-D-R-NH2) labeled with MCA.
Cell lysates or other samples that contain cathepsin-D will cleave the synthetic substrate to release fluorescence, which can then easily be quantified using a fluorometer or fluorescence plate reader at Ex/Em = 328/460 nm.
This product is manufactured by BioVision, an Abcam company and was previously called K143 Cathepsin D Activity Fluorometric Assay Kit. K143-100 is the same size as the 100 test size of ab65302.
Apoptosis can be mediated by mechanisms other than the traditional caspase-mediated cleavage cascade. There is growing recognition that alternative proteolytic enzymes such as the lysosomal cathepsin proteases may initiate or propagate proapoptotic signals. Cathepsins are lysosomal enzymes that are also used as sensitive markers in various toxicological investigations.
Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.
Storage instructionsStore at -20°C. Please refer to protocols.
Components Identifier 2000 tests 100 tests 100 tests 2000 tests CD Cell Lysis Buffer WM 20 x 100ml 1 x 100ml 1 x 100ml 20 x 100ml CD Reaction Buffer NM 20 x 5ml 1 x 5ml 1 x 5ml 20 x 5ml Substrate II Brown 20 x 200µl 1 x 200µl 1 x 200µl 20 x 200µl
RelevanceCathepsin D is a normal lysosomal protease that is expressed in all cells. It is an aspartyl protease with a pH optimum in the range of 3-5, and contains two N linked oligosaccharides. Cathepsin D is synthesized as an inactive pro enzyme. Activation involves the proteolytic removal of the 43 amino acid profragment and an internal cleavage to generate the two chain form made up of 34 and 14 kDa subunits. Cathepsin D contains the mannose-6-phosphate lysosomal localization signal that targets the enzyme to the lysosomal compartment where it functions in the normal degradation of proteins. In certain tumor cells, Cathepsin D is abnormally processed and is secreted in its precursor form. Numerous clinical studies as well as in vitro evidence suggest that cathepsin D plays an important role in malignant transformation and may be a useful prognostic indicator for breast cancer and possibly Alzheimer's disease.
Cellular localizationLysosome. Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
- Lysosomal aspartyl protease
Enzyme activity of Cathepsin B and Cathepsin D was determined using Cathepsin B/D activity assay kit (ab65300 and ab65302). Neuro2a cell lysates were centrifuged at 10,000 xg for 10 minutes at 4ºC and supernatant was used for the assay. Cathepsin activity was performed at the 24 and 48 hours time points by cleavage of the fluorescence peptide substrate [DnP-DR-MCA, GKPILFFRLK(DnP)-DR substrate peptide labeled with MCA]. Data represent the mean ± SD (*p<0.05, **p<0.001).
Cathepsin D levels were measured in standard control samples from ab119586; background signal subtracted (duplicates +/- SD).
Cathepsin D measured in mouse tissue lysates (mg of extracted protein), background signal subtracted (duplicates +/- SD).
Cathepsin D measured in cell lysates, background signal subtracted (duplicates +/- SD).
Cathepsin D measured in Jurkat cells.
ab65302 has been referenced in 115 publications.
- Stahl-Meyer J et al. Lysosomal Changes in Mitosis. Cells 11:N/A (2022). PubMed: 35269496
- Dettleff P et al. Molecular Characterization of Embryos with Different Buoyancy Levels in the Yellowtail Kingfish (Seriola lalandi). Animals (Basel) 12:N/A (2022). PubMed: 35327117
- Xie YX et al. Lysosomal exocytosis releases pathogenic a-synuclein species from neurons in synucleinopathy models. Nat Commun 13:4918 (2022). PubMed: 35995799
- Li GN et al. Elaiophylin triggers paraptosis and preferentially kills ovarian cancer drug-resistant cells by inducing MAPK hyperactivation. Signal Transduct Target Ther 7:317 (2022). PubMed: 36097006
- Navarro-Romero A et al. Lysosomal lipid alterations caused by glucocerebrosidase deficiency promote lysosomal dysfunction, chaperone-mediated-autophagy deficiency, and alpha-synuclein pathology. NPJ Parkinsons Dis 8:126 (2022). PubMed: 36202848