Product Details
| abID | ab126779 |
|---|---|
| Cat # +Size | K148-100 |
| Size | 100 assays |
| Detection Method | Fluorescence (Ex/Em = 328/460 nm) |
| Applications | Assay kit to detect putative Cathepsin D inhibitors |
| Features & Benefits | • Simple one-step procedure; takes only 1 hours • Fast, robust and convenient • Cathepsin-D inhibitor screening assay is a fast and robust assay to screen potential inhibitors • Positive control inhibitor, Pepstatin A is included in the kit |
| Kit Components | • CD Reaction Buffer • CD Substrate (1 mM) • Cathepsin D, human (lyophilized) • Pepstatin A (1mM in DMSO) |
| Storage Conditions | -80°C |
| Shipping Conditions | Dry Ice |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
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. The Cathepsin D Activity Assay 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 = 328/460 nm. The relative efficacy of test inhibitors are compared to the positive control inhibitor, Pepstatin A (IC50 < 0.1 nM). The Cathepsin D assay is simple, straightforward, and can be adapted to 96-well plate assays and is suitable for high throughput screening (HTS).
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How should tissue lysates be prepared?
We have never tested this kit on tissue samples. However, if you want to test, you could homogenize the tissue in Cell Lysis buffer, preferably using a Dounce homogenizer. Centrifuge the lysate and use the supernatant for the assay.
Will this kit work with mouse or rat samples?
This kit is compatible with all mammalian samples including mouse and rat.
Can we interchange the buffer from this kit with K143?
Each buffer is of different composition and they cannot be used
Can the kit work on bacteria or yeast cells?
The kit has been standardized for mammalian cells only
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact yopur regional BioVision distributor.
What is the shelf life of this kit?
The expiry date is typically 1 year from the date of shipment
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Are Cathepsin D substrates used in K143 and K148 the same or different? Can I use K148 substrate in K143?
The substrates in K143 and K148 are the same. Therefore, the you can use K148 substrate in K143.
Jessica L. Schwartz-Roberts et al., GX15-070 (Obatoclax) Induces Apoptosis and Inhibits Cathepsin D- and L–Mediated Autophagosomal Lysis in Antiestrogen-Resistant Breast Cancer Cells. Mol. Cancer Ther., Apr 2013; 12: 448 - 459.
For more citations of this product click here
