Key features and details
- Assay type: Quantitative
- Detection method: Fluorescent
- Platform: Microplate reader
- Assay time: 2 hr
- Sample type: Cell Lysate, Tissue Extracts
Product nameCathepsin B Activity Assay Kit (Fluorometric)
See all Cathepsin B kits
Sample typeTissue Extracts, Cell Lysate
Assay time2h 00m
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 at Ex/Em 400/505 nm.
The Cathepsin B assay protocol is simple, straightforward, and can be adapted to 96-well plate assays. Assay conditions have been optimized to obtain the maximal activity.
Cathepsin B assay protocol summary:
- add samples to wells
- add reaction mix and incubate for 1-2 hr at 37ºC
- analyze with microplate reader
Storage instructionsStore 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
RelevanceCathepsin 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 localizationLysosome. Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
- Amyloid precursor protein secretase
- APP secretase
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.
ab65300 has been referenced in 47 publications.
- Shen S et al. BRD4 Inhibition Protects Against Acute Pancreatitis Through Restoring Impaired Autophagic Flux. Front Pharmacol 11:618 (2020). PubMed: 32457617
- 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
- Banday AA & Lokhandwala MF Renal Dopamine Oxidation and Inflammation in High Salt Fed Rats. J Am Heart Assoc 9:e014977 (2020). PubMed: 31880979
- Yun HR et al. Palmitoyl Protein Thioesterase 1 Is Essential for Myogenic Autophagy of C2C12 Skeletal Myoblast. Front Physiol 11:569221 (2020). PubMed: 33178040
- Wu Y et al. Regulation of endo-lysosomal pathway and autophagic flux by broad-spectrum antipathogen inhibitor ABMA. FEBS J 287:3184-3199 (2020). PubMed: 31901207