Recently, the non-FRET EnSens technology was launched for  in vitro assessment of specific protease activities (Enzium, Inc.). Up to date, the EnSens substates were validated for the detection of protease activity in microplate assays. Now, Enzium has extended the applicability of their Ensens method to 3 D live-cell imaging. On the top of this, they announce that the experimental procedure is easy and non-toxic for cell cultures and co-cultures. So how does live-cell imaging help you locate protease activities in in vitro tumor invasion assays ?

How can you overcome the limits of FRET based protease activity assays for MMPs, ADAMs, Thrombin, Furin and Factor Xa?

Usually screenings for inhibitors of diverse protease activities are conducted with FRET or FRET-like assays, in which the protease substrate has to be coupled to a fluorophore and a quencher.

These assays use relatively short peptide substrates and often only the core of the specific protease recognition site can be exposed to the protease of interest. This can result in a number of false positives and negatives because the recognition site might not be highly selective. Furthermore these FRET substrates often show limited stability.

Fluorophores commonly used are furthermore characterized by a low level of photo stability, and tend to have short wavelengths which can result in higher backgrounds due to interfering fluorescence. Thus the signal-to-noise ration is sometimes on the borderline.

Another challenge with FRET based and FRET-like assays can be a high level of pH sensitivity and a low solubility of the substrates in aqueous solution. So, what’s the solution?