Authors
Dimitrios Vlachakis, Andrea Brancale, Colin Berry, and Sophia Kossida
Summary
A new assay for the measurement of helicase enzyme activity was developed for the evaluation of the potency of potential inhibitors. This assay involves the use of a DNA or RNA duplex substrate and recombinant purified helicase. The DNA duplex consists of a pair of oligonucleotides, one of which is biotinylated and the other is digoxygenin (DIG)-labelled, both at their respective 5’ termini. This DNA duplex is immobilised, via the biotin molecule, on the surface of a neutravidin-coated 96 well plate. Helicase activity results in DNA unwinding upon activation by ATP, leading to the release of the DIG labelled oligonucleotides, which translates in signal (luminescence) reduction with respect to control wells. This signal can be produced and quantified with the aid of a chemiluminescence antibody.Further details
The protocol was published on Protocol Exchange on 22 December 2011. To see the entire protocol, click on the source link.Advertisement
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