Cathepsin B Activity Assay Kit (Fluorometric) (ab65300)
Key features and details
- Assay type: Quantitative
- Detection method: Fluorescent
- Platform: Microplate reader
- Assay time: 2 hr
- Sample type: Cell Lysate, Tissue Extracts
Overview
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Product name
Cathepsin B Activity Assay Kit (Fluorometric)
See all Cathepsin B kits -
Detection method
Fluorescent -
Sample type
Tissue Extracts, Cell Lysate -
Assay type
Quantitative -
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 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 -
Platform
Microplate reader
Properties
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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
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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
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Images
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Functional studies - Cathespin B Activity Assay Kit Bong G.J., PLoS One 8(10), Fig 5F. doi:10.1371/journal.pone.0076466 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/
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).
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Functional assays: Cathepsin B Activity Assay Kit (Fluorometric) (ab65300)Cathepsin B activity in HL60 cell lysates (2.5x10e6 cells) with the addition of inhibitor.
