Pull down of purified, bacterially expressed PP1 isoforms via His-tagged RNA Helicase Ddx21.

Abstract

<p>Bacterially expressed and purified PP1 (α, β, γ) and His6-Ddx21 (wt, motif1, motif2, double) were incubated together and then mixed with precleared Ni-NTA beads, all at 4°C. Each PP1 isoform was also incubated without Ddx21 (-) to verify unspecific binding of PP1 to the Ni-NTA beads. Enriched His6-Ddx21 and co-precipitated PP1 isoforms were washed and boiled in 2 X SDS-PAGE sample buffers. PP1 inputs, Ddx21 eluates and negative control (-) eluates were separated and analysed by western blot for the presence of Ddx21 and PP1.</p