What will the Next-Gen Protease activity detection be?

Fluorescent technologies enable the measurement of protease activities and the screening of compounds influencing protease activities. Fluorescence Resonance Energy Transfer (FRET) but also TR-FRET, Q-FRET together with Time Resolved Fluorescence (TRF) and Fluorescence Lifetime (FLT) are popular assays, in which protease substrates occupy a central place. These assays are based on the specific recognition and cleavage of a peptide sequence (substrate) by the protease of interest. If the substrate is not optimally designed (or too short), experimental output can be unrelevant: low selectivity and specificity, high background, false negatives or positives…). In addition, the data can also be affected by the reaction buffer (pH, compound solvent…).This lack of relevant biological information is at the origin the emergence of “Next-Gen” fluorescent protease substrates.

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Direct localization of MMP activity in 3D tumor invasion model

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 ?

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MMP-25 activity assay – the first one on the market!

With the recent release of the first commercially available and ready-to-use assay to detect MMP-25 activity , Enzium has opened a new era for screening protease activities through a robust non-FRET experimental approach. A good opportunity to take a look at the EnSens technology.

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EnSens, or how to overcome FRET assay limitations

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? [Read more…]