Cathepsin D Activity Assay Kit (Fluorometric) (ab65302)


  • Product name
    Cathepsin D Activity Assay Kit (Fluorometric)
    See all Cathepsin D kits
  • Sample type
    Tissue Extracts, Cell Lysate
  • Assay type
  • Assay time
    2h 00m
  • Species reactivity
    Reacts with: Mouse, Human
    Predicted to work with: Mammal
  • Product overview

    Abcam's Cathepsin D Activity Assay Kit 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.
    Visit our FAQs page for tips and troubleshooting.

  • Notes

    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.

  • Tested applications
    Suitable for: Functional Studiesmore details


  • Storage instructions
    Store 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
  • Research areas
  • Relevance
    Cathepsin 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 localization
    Lysosome. Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
  • Alternative names
    • CLN10
    • CPSD
    • CTSD
    • Lysosomal aspartyl protease
  • Database links

Associated products


Our Abpromise guarantee covers the use of ab65302 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
Functional Studies Use at an assay dependent dilution.


  • 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, n = 3).

  • 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).



This product has been referenced in:
  • Zou ZZ  et al. Synergistic induction of apoptosis by salinomycin and gefitinib through lysosomal and mitochondrial dependent pathway overcomes gefitinib resistance in colorectal cancer. Oncotarget 8:22414-22432 (2017). Functional Studies ; Human . Read more (PubMed: 26461472) »
  • Jinn S  et al. TMEM175 deficiency impairs lysosomal and mitochondrial function and increases a-synuclein aggregation. Proc Natl Acad Sci U S A 114:2389-2394 (2017). Read more (PubMed: 28193887) »

See all 15 Publications for this product

Customer reviews and Q&As

The “Cathepsin D Activity Assay Kit (fluorometric)” (ab65302) was used to determine Cathepsin D action in cell culture media of four breast cell lines (MCF 10A, MCF-7, MDA MB231, T47D). Since the product data sheet shows the procedure to determine the enzyme activity in cellular homogenates and tissues but not in cell cultures, we modified the protocol suggested by the manufacturers as specified below.
Note: We call “maintenance medium” to the medium that has not been incubated with cells and “conditioned medium” to the medium taken from cultured cells.

- Cathepsin D enzymatic activity measurement protocol for cell culture media:
1- The four cell lines conditioned mediums were collected and centrifuged for 10 minutes at 5000xg at 4°C.
2- 50µl of each supernatant sample were transferred to a 96 black well plate.
3- 52 µl of the Reaction Mix (50µl reaction buffer + 2µl substrate) were added to each well.
4- The plate was protected from light and incubated at 37°C for 2 hours.
5- The output was measured on the microplate reader “Fluoroskan Ascent FL. Thermo Electron Corporation” (λEx/Em = 328/460).

- The results are shown in the attached Table 1.
- Considering the results, the kit was not found useful to measure Cathepsin D activity in cell culture media. The reason could be due to a potential auto fluorescence of the cell culture media (with or without phenol red).

Abcam user community

Verified customer

Submitted Nov 19 2015

CathepsinD activity in yeast

Good Excellent 5/5 (Ease of Use)
This kit works well to quantify the activity of yeast Cathepsin D (Pep4) in Saccharomyces cerevisiae. The fluorometric measurement has been performed as described in the protocol attached to the kit, with one small modification: cell lysis with supplemented lysis buffer was not efficient for yeast due to the cell wall, so a mechanical disruption step using glas beads in supplemented lysis buffer was added.

Abcam user community

Verified customer

Submitted Feb 19 2015

We do not recommend to prepare one lysate to use with ab65302, ab65306 and ab65307.
It is important to prepare separate lysates for each kit, using the specific lysis buffer provided. This is because the cathepsins have very specific pH and other c...

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Cathepsin D Activity Assay

Excellent Excellent 5/5 (Ease of Use)
Very easy to use, worked well on both SH-SY5Y cells and freshly prepared human tissue. Loaded 200 micrograms / ml of total protein for SH-SY5Y cells, and 1mg/ml total human cortical protein.

Abcam user community

Verified customer

Submitted Feb 24 2014

Es preferible utilizar un pellet celular fresco, en lugar de uno congelado. De cualquier manera, si el pellet se ha almacenado a -80C inmediatamente después de lisar las células en principio no hay problema en utilizarlo con este kit. Hay...

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1) We have not tested adding increasing concentrations of DMSO with this kit.

I would definitely recommend adding protease inhibitor cocktail instead of DMSO, such as ab65621
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Thank you for contacting us.

We have used this assay only with cell and tissue lysates, which will be lysed in the lysis buffer. To use with body fluid, I would recommend diluting it with the lysis buffer. The PBS should not interfere with the...

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Thank you for contacting us.

The fluorescent compound in this kit is MCA which has absorbance maxima ˜350 and excitation maxima ˜460. Please click the link for details (Fig 1)

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Thank you for your inquiry.

I can confirm that an inhibitor would be a good negative control for your customer. But it is also not necessary to to have a negative control for every sample.

This kit is not measuring absolute activity...

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Thank you for contacting Abcam.

We have not tested any of the kits with fish samples but theoretically as long as there are active enzymes in the sample, the kit should detect it. Fish cell lysates can be prepared as for the mammalian cells a...

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1-10 of 17 Abreviews or Q&A


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