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Fluorescence anisotropy assay for the traceless kinetic analysis of protein digestion.

Analytical chemistry (2008-04-25)
Felix Cleemann, Peter Karuso
ABSTRACT

A novel fluorescence polarization assay based on the natural fluorophore epicocconone has been developed. This assay allows the rapid and accurate determination of enzyme kinetic parameters as well as inhibition constants through the measurement of fluorescence anisotropy on the actual substrate of the protease. It takes advantage of epicocconone's ability to reversibly react with proteins to form an internal charge-transfer complex that is highly fluorescent. The protein-substrate is labeled in situ without the need for prior incubation and/or derivatization steps, which saves time and effort compared to methods employing specifically labeled protein-substrates. The assay can be carried out in 96- or 384-well plates, making it suitable for high-throughput applications in drug development and biotechnology.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
α-Chymotrypsin from bovine pancreas, Type II, lyophilized powder, ≥40 units/mg protein
Sigma-Aldrich
Elastin, soluble from bovine neck ligament, salt-free, lyophilized powder