The Caspase-3/CPP32 Fluorometric Protease Assay Kit from BioVision is designed to detect the specific enzymatic activation of Caspase-3. In addition to their physiological targets, caspases cleave artificial substrates consisting of an appropriate sequence of, generally, four amino acids. Caspases cleave substrata on the C-terminal side of an aspartic acid residue that can be bound to a fluorogenic or chromogenic amine. The BioVision Caspase-3 Fluorometric Assay Kit exploits this activity by using a fluorogenic substrate AFC (7-amino-4-trifluoromethyl coumarin). Since fluorometric analysis is highly sensitive, this assay is suitable for measuring even low amounts of enzymatic activity. Samples can be read in a fluorometer or a fluorescence microtiter plate reader equipped with a 400-nm excitation filter and 505-nm emission filter. In addition, the entire fluorometric enzymatic assay can be performed directly in a 96-well plate, saving reagents and samples. Comparison of the fluorescence of AFC from an apoptotic sample with that of an uninduced control allows determination of the fold increase in caspase-3 activity. The kit comes with enough reagents for 25, 100, 200 or 400 assays, depending on which kit is ordered (we used the kit for 100 assays). The kit comes with all of the components necessary to perform the activity assay.
The Caspase-3 Fluorometric Protease Assay Kit is a great tool for determining whether Caspase-3 is activated in a given experiment. Caspase-3, sometimes called the “executioner caspase”, serves as a positive indicator of apoptosis. This kit has both technical and economical advantages. Not only can it be complementary to Western blotting, which, in turn, reveals caspase activation triggered by their cleavage, but also it can be fundamental when Western blotting is particularly problematic. In addition, the BioVision product is economically competitive versus both other analogous products and other scientific approaches to the problem, such as Western blotting. Often, immunoblotting can be expensive due to high antibody costs. Although it is possible to buy single caspase-specific fluorometric reagents separately from BioVision, it is economically convenient to buy the entire kit when one wishes to perform an initial screen for Caspase-3 activity.
In my experience, the results obtained from using this kit could be confirmed by Western blotting with a cleaved Caspase-3 antibody. But there are a few issues one should keep in mind. First of all, one should use fresh lysates for the assay and keep all reagents on ice. I found the results to be highly variable when thawed lysates were used. Secondly, one must work quickly since Caspase- 3 activity was found to decline the longer the assay lasted. The Caspase-3 Fluorometric Protease Assay Kit comes from the manufacturer with a series of stock solutions; this offers a few advantages. First, purchase of the kit offers the savings that result from buying the other materials required separately. Secondly, using the kit offers the freedom of adjusting the experimental conditions for the assay. One should note, that assay conditions must be carefully set, including appropriate positive and negative controls to check specificity. Since specificity is also affected by the substrate concentration, this is a critical issue. The manufacturer recommends a final concentration of 50 uM of Caspase-3, which I found to be effective.
In summary, the BioVision Caspase-3 Fluorometric Protease Assay Kit is innovative, decidedly reliable and has a competitive price with respect to similar products. Therefore, I recommended it.
Jennifer L. Seachrist, Ph.D.