The Caspase Fluorometric Substrate Set from BioVision is a reagent set designed to detect specific caspases by their enzymatic activities. The set includes reagents to screen for 7 members of the caspase family described so far (including caspase-1, -2, -3, -5, -6, -8 and -9). 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 residue that can be bound to a fluorogenic or chromogenic amine. The BioVision Caspase Fluorometric Substrate Set exploits this activity by using a fluorogenic substrate AFC (7-amino-4-trifluoromethyl coumarin), which has an excitation-emission wavelenght pair of 400-505 nm, respectively. Because fluorometric analysis is highly sensitive, this assay is suitable for measuring even low amounts of enzymatic activity. In addition, the fluorometric enzymatic assay can be carried out in 96-well plates saving reagents and samples.
The Caspase Fluorometric Substrate Set is a great tool for determining which specific caspases are involved in a given biological phenomenon for both technical and economical reasons. 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 (e.g. in our experience most commercially available antibodies do not work well to detect murine caspases by Western blot). In addition, the BioVision product is economically competitive versus both other analogous products and other scientific approaches to the problem (e.g. Western blotting, where the cost of antibodies can add up). Although it is possible to buy single caspase-specific fluorometric reagents separately from BioVision, there is a great economical convenience to buy the entire set when a first screening is required.
The Caspase Fluorometric Substrate Set comes from the manufacturer as a set of 7 different chemicals in a highly concentrated (1 mM) stock solution. This “kit” is sufficient for at least 25 tests and the fact that it comes as a series of stock solutions has a few advantages. This includes the savings that result from buying the other materials required separately (in contrast to purchasing all inclusive kits which are usually expensive) and the freedom of determining the experimental conditions for the assay.
About the latter point, it is worth noting that when a peptide is referred to as "caspase-X specific" it means that the substrate is cleaved by caspase-X more efficiently, but not exclusively, by caspsase-X. Therefore, 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. In my experience, a substrate-AFC concentration lower than those suggested by the manufacturer can be used, with the additional advantage of a longer lasting product.
In general, the BioVision Caspase Fluorometric Substrate Set is technically innovative, highly reliable and has a price very competitive in respect to analogous products. Therefore, it is enthusiastically recommended.
Dario Coletti, PhD
Post-Doctoral Fellow
Mount Sinai School of Medicine