Lapik, Y.R., Misra, J.M., Lau, L.F. and Pestov, D.G.
Notes: The authors show that substitution of alanine for the conserved glycine 224 of Nog1, a eukaryotic GTPase, disrupts assembly of pre-60S ribosome subunits but does not significantly affect GTP binding. Amino acids 1–357 of Nog1 (Nog1NG) were expressed with an N-terminal biotinylated tag in E. coli, then purified using the SoftLink™ Soft Release Avidin Resin. The resin was incubated with the cleared lysate for 2 hours with mixing and washed with B300 buffer (25mM Tris-HCl [pH7.4], 10mM MgOAc2, 10% glycerol, 0.05% Brij 30, 1mM dithiothreitol, 300mM KOAc) and B1000 buffer (25mM Tris-HCl [pH7.4], 10mM MgOAc2, 10% glycerol, 0.05% Brij 30, 1mM dithiothreitol, 1M KOAc). Purified protein was eluted with B300 buffer containing 5mM biotin, and the protein was concentrated and biotin removed by ultracentrifugation. The protein was estimated to be 95% pure by SDS-PAGE. GTP-binding assays were performed by UV cross-linking purified Nog1NG and [α-32P]-GTP, recovering the protein by binding to SAM2 Biotin Capture Membrane and quantifying the amount of GTP bound using a Phosphorimager. (3804)