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.
Sample Type:
Purified Cathepsin D, Recombinant Cathepsin D
Intended Use:
Assay kit to detect putative Cathepsin D inhibitors.
Test Principle:
The Cathepsin D Activity BioAssay(TM) kit is a fluorescence-based assay that utilizes the preferred cathepsin D substrate sequence GKPILFFRLK(Dnp)-D-R-NH2) labeled with MCA. Cathepsin D will cleave the synthetic substrate to release the quenched fluorescent group MCA, which can then easily be measured using a fluorometer or fluorescence plate reader at Ex/Em = 328/460nm. The relative efficacy of test inhibitors are compared to the positive control inhibitor, Pepstatin A (IC50 < 0.1nm). The Cathepsin D assay is simple, straightforward, and can be adapted to 96-well plate assays and is suitable for high throughput screening (HTS).
Kit Components:
CD Reaction Buffer, 1x10ml
CD Substrate, 1mM, 1x0.2ml
Cathepsin D, human, lyophilized, 1x vial
Pepstatin A, 1mM in DMSO, 1x20ul
Storage and Stability:
Store powder at 4 degrees C liquid at -20 degrees C. Store other components at 4 degrees C. Stable for at least 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
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