Enzyme assays are rarely optimized for both biological (e.g., cell lysates) and in vitro (e.g., recombinant enzyme) samples. The availability of an assay that can be used for both types of samples is advantageous because it allows evaluation of activators or inhibitors with native enzyme from biological fluids. With proteases, development of a specific and versatile assay is difficult because many different proteases with similar substrate specificity are present in biological samples. For example, substrates for cathepsin S are clipped with a similar efficiency by cathepsin L, B, and K, and to distinguish these proteases through with a soluble assay is very difficult.

InnoZyme™ Protease Assays utilize two different methodologies to overcome these difficulties. In the first, enzyme immunocapture is used to specifically isolate the protease before the activity measurement. The second type of assay is homogenous and does not employ enzyme immunocapture, but includes a selective protease inhibitor to monitor the activity of a particular protease. For the immunocapture assays, an antibody that specifically captures the protease immobilized to the wells of a 96-well plate (Figure 1). After the sample is added, a washing step is performed, and then an internally quenched fluorescent substrate is added. This type of substrate is also used for the homogenous InnoZyme assays. An increase in fluorescence results when the substrate is cleaved by the protease, separating the fluorophore and the quencher. The level of fluorescence is directly related to enzyme activity, with typical detection ranges between 3 and 100 ng/ml. All assays display superb specificity. For example, the InnoZyme Cathepsin S assay has a cross reactivity with cathepsin B, L, and K of less than 1%. Inhibitor or activator screening can be performed with biological samples, or by using the recombinant control that is provided. InnoZyme Protease Assays are well suited for the specific and sensitive measurement of activity of individual proteases in complex biological samples, and thus are superb tools for studying proteases involved in apoptosis, angiogenesis, and inflammation.

• Highly selective: Exquisite specificity through protease immunocapture or selective inhibition
• Versatile: Sensitive detection and measurement of native protease activity
• Useful: Screen effects of inhibitors or activators
• Convenient: 96-well format, fluorometric, non-radioactive detection