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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.
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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.
|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|
Our Abpromise guarantee covers the use of ab65300 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|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).
Cathepsin B activity in HL60 cell lysates (2.5x10e6 cells) with the addition of inhibitor. Background signal subtracted, duplicates; +/- SD.
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