Purification of rHuR and optimization of the AlphaScreen assay.
A) Representative 15% SDS-PAGE and Coomassie staining of purified rHuR protein (80 ng) recovered after ZebaTM Spin Desalting Columns dialyzation and western blot on the same sample (20 ng) using a polyclonal anti-HuR antibody. B) Bi-TNF RNA probe and rHuR protein double titration to determine optimal ligand concentrations with the AlphaScreen anti-c-Myc-Acceptor and streptavidin-Donor beads of the c-Myc detection kit (PerkinElmer), resulting in 1 nM and 50 nM for rHuR and Bi-TNF, respectively. C) Bi-TNF titration with 1 nM of rHuR. “Hooking effect” is shown for concentrations over 50 nM of RNA ligand (as the point at 100 nM in the log scale). Mean and standard deviation values derive from four independent experiments with four rHuR protein preparations.
