• Product name
    Cathepsin B Activity Assay Kit (Fluorometric)
    See all Cathepsin B kits
  • Sample type
    Tissue Extracts, Cell Lysate
  • Assay type
  • Assay time
    2h 00m
  • Product overview

    Cathepsin B Activity Assay kit (Fluorometric) (ab65300) is a fluorescence-based assay that utilizes the preferred cathepsin-B substrate sequence RR labeled with AFC (amino-4-trifluoromethyl coumarin). Cell lysates or other samples that contain cathepsin-B will cleave the synthetic substrate RR-AFC to release free AFC. The released AFC can easily be quantified using a fluorometer or fluorescence plate reader. The Cathepsin B assay is simple, straightforward, and can be adapted to 96-well plate assays. Assay conditions have been optimized to obtain the maximal activity.
    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.


  • Storage instructions
    Store at -20°C. Please refer to protocols.
  • Components Identifier 100 tests
    CB Cell Lysis Buffer WM 1 x 25ml
    CB Inhibitor (1mM) Red 1 x 20µl
    CB Reaction Buffer NM 1 x 5ml
    Substrate Ac-RR-AFC (10mM) Brown 1 x 200µl
  • Research areas
  • Relevance
    Cathepsin B is a normal lysosomal proteinase that is expressed in all cells. It is a cysteine endoproteinase structurally and functionally related to the papain family of proteases. Cathepsin B is synthesized as an inactive pro enzyme which, by removal of a 62 amino acid propeptide, is activated to the single chain form of 31 kDa or the two chain form (25 and 5 kDa subunits). Matured Cathepsin B is normally localized in the lysosomes where it functions in protein turnover. Cathepsin B is also shown capable of degrading extracellular matrix proteins at acidic and neutral pH. Endogenous inhibitors of cysteine proteases, the cystatins and stefins, may play a major role in regulating Cathepsin B activity. In tumor cells and in cells exposed to mitogens, cathepsin B displays altered cellular trafficking resulting in the secretion of the 43 kDa precursor. Elevated levels of Cathepsin B correlate with malignancy suggesting that this enzyme may be a useful prognostic marker for several types of human cancers. Cathepsin B has also been implicated in the pathogenesis of rhematoid arthritis, muscular dystrophy and tumour metastasis. It is also known as amyloid precursor protein secretase and is involved in the proteolytic processing of amyloid precursor protein (APP).
  • Cellular localization
    Lysosome. Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
  • Alternative names
    • Amyloid precursor protein secretase
    • APP secretase
    • APPS
    • Cathepsin B1
    • CathepsinB
    • CPSB
    • CTSB
    • Preprocathepsin B
    see all


  • 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 B activity in HL60 cell lysates (2.5x10e6 cells) with the addition of inhibitor. Background signal subtracted, duplicates; +/- SD.



This product has been referenced in:
  • Wang Y  et al. Cathepsin B aggravates coxsackievirus B3-induced myocarditis through activating the inflammasome and promoting pyroptosis. PLoS Pathog 14:e1006872 (2018). Functional Studies . Read more (PubMed: 29360865) »
  • Tian L  et al. The cytotoxicity of coxsackievirus B3 is associated with a blockage of autophagic flux mediated by reduced syntaxin 17 expression. Cell Death Dis 9:242 (2018). Read more (PubMed: 29445155) »

See all 20 Publications for this product

Customer reviews and Q&As

Thank you for your enquiry. It may be possible to use plasma samples with this kit plasma. To date, however, we have no data testing plasma samples with this kit. If you do decide to use ab65300 with plasma samples I'd encourage you to submit an Abrevi...

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I'm sorry we cannot share the composition of the lysis or the reaction buffer due to proprietary reasons. However, the lysis buffer contains Tween 20 which might not always work well with all enzyme-based assays.

Thank you for contacting us.

The sensitivity of these cathepsins is: 10 – 100 ng/assay.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

The lab let me know that they do not have any additional tips for tissue processing other than the ones we mention on the FAQ site.

12. How should tissue be prepared for the kits?

Samples should be completely homogenized, pr...

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Free AFC is available as product ID ab129872.

Thank you for your enquiry.

I can confirm this ab65300 Cathepsin B Activity Assay Kit (Fluorometric) is compatible with all mammalian samples, so it can be used with mice and rat samples. Here are a few citations which I hope may be benefici...

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Thank you for contacting us.

I have had the chance to speak with the lab regarding your question. Each buffer is of different composition and they cannot be used interchangeably.

I hope this information is helpful to you. Plea...

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Thank you for contacting us and your interest in our products.

The Cathepsin B Activity Fluorometric Assay Kit (ab65300) has not currently been tested with plasma samples.However, as long as theplasma samples contain Cathepsin B within the de...

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According to our records, ab65300 was proving difficult to use and we were in contact in order to help resolve the issue. Looking at our correspondence, it appears that we are awaiting more details in order to help us better understand the diff...

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Thanks for your call today and for letting us know about the problem with ab65300. As we discussed, please send your data and I will forward it on to the lab to see if there is any explanation or suggestion for your results. Could you also tell ...

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1-10 of 12 Abreviews or Q&A


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