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.
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 100 tests CD Cell Lysis Buffer WM 1 x 25ml CD Reaction Buffer NM 1 x 5ml CD Substrate (1mM) Brown 1 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 67 publications.
- Wang J et al. Betalain exerts a protective effect against glaucoma is majorly through the association of inflammatory cytokines. AMB Express 10:125 (2020). PubMed: 32666339
- Kobayashi S et al. Hyperglycemia-induced cardiomyocyte death is mediated by lysosomal membrane injury and aberrant expression of cathepsin D. Biochem Biophys Res Commun 523:239-245 (2020). PubMed: 31862139
- Zhang X et al. Ischemia-induced upregulation of autophagy preludes dysfunctional lysosomal storage and associated synaptic impairments in neurons. Autophagy N/A:1-24 (2020). PubMed: 33111641
- Banday AA & Lokhandwala MF Renal Dopamine Oxidation and Inflammation in High Salt Fed Rats. J Am Heart Assoc 9:e014977 (2020). PubMed: 31880979
- Zeng XJ et al. A2A R inhibition in alleviating spatial recognition memory impairment after TBI is associated with improvement in autophagic flux in RSC. J Cell Mol Med 24:7000-7014 (2020). PubMed: 32394486