PMC:2728203 / 26013-27018 JSONTXT

{"project":"2_test","denotations":[{"id":"18190927-15208322-62517995","span":{"begin":125,"end":127},"obj":"15208322"},{"id":"18190927-8918934-62517996","span":{"begin":206,"end":208},"obj":"8918934"}],"text":"Purified recombinant His-tagged Gfd1 was prepared as previously described to examine the interaction between Nab2-N and Gfd1.27 The Gfd1 protein was attached to CNBr Sepharose beads as previously described.38 Briefly, CNBr Sepharose beads (Amersham Pharmacia Biotech) were swollen and washed in 1 mM HCl. Beads were transferred to coupling buffer (100 mM NaHCO3, pH 8.3, and 500 mM NaCl) and added to 2–5 mg of Gfd1 in coupling buffer. Coupling was carried out at 4 °C overnight. Residual active groups were blocked with 1 M Tris–HCl, pH 8.0, for 2 h at room temperature. Beads were then washed successively and extensively four times in coupling buffer and acid wash buffer (0.1 M sodium acetate, pH 4.0, and 500 mM NaCl). For binding assays, 10 μg of Nab2-N was incubated with 50 μl of Gfd1 beads for 2 h at 4 °C. Beads were then washed twice in PBS, and bound proteins were eluted with 100 μl of sample buffer. Samples were resolved by PAGE, and bound proteins were detected by Coomassie Blue staining."}