Biochemical characterization of human upf1 helicase

Abstract

We present here the biochemical characterization of human Upf1 helicase core (hUpf1c). hUpf1c is overexpressed as a GST fusion protein in Escherichia coli and purified using chromatographic methods. In vitro ATP binding and single-stranded RNA (ssRNA) binding activities are measured using dot-blot technique. Measurement of RNA-dependent ATPase activity is performed by thin layer chromatography (TLC). The ATP-modulated ssRNA binding activity is examined by surface plasma resonance (SPR). The binding of double-stranded DNA (dsDNA) to hUpf1c is checked by electrophoretic mobility shift assay (EMSA, gel shift assay). © 2009 Humana Press, a part of Springer Science+Business Media, LLC.