Sulfolink resin was charged with cysteine as described earlier (26) . Yeast cells were grown in YPAD media to mid log phase, collected and washed with binding buffer (10 mM Tris-HCl, pH 7.5; 5 mM MgCl 2; 60 mM NH 4 Cl; 2 mM DTT). Cells were suspended in binding buffer and disrupted using a Mini Bead Beater. Lysates were centrifuged at 30 000g for 30 min in Beckman MLS 50 rotor. Supernatant (2 ml) was removed and added to 2 ml of cystein-charged Sulfolink slurry (50% resin equilibrated with binding buffer) and incubated on ice for 15 min with mixing as resin sediments. After incubation resin was spun down at 1500g for 0.5 min. Supernatants were removed and resin washed five times with 5 ml of binding buffer. After washing, resin was suspended in 1 ml of elution buffer (10 mM Tris-HCl, pH 7.5; 10 mM MgCl 2; 0.5 M KCl; 1 mg/ml heparin; 2 mM DTT) and incubated for 5 min on ice with occasional mixing. The suspensions were centrifuged, supernatant collected, and elution was repeated two more times. Supernatants were combined (3 ml total volume) and GTP and pH neutralized puromycin were added to 1 mM each. After incubation at 30 C for 30 min, reaction mixtures were loaded on top of a 1 ml glycerol cushion (10 mM Tris-HCl, pH 7.5; 10 mM MgCl 2; 0.5 M KCl; 2 mM DTT; 25% glycerol) and centrifuged at 100 000g for 16 h. Ribosome pellets were suspended in 2 ml of elution buffer without heparin, loaded on top of 2 ml glycerol cushions and centrifuged at 100 000g for 16 h. Ribosome pellets were resuspended in storage buffer [50 mM HEPES-KOH pH 7.6; 5 mM Mg(CH 3 COO) 2; 50 mM NH 4 Cl; 1 mM DTT; 25% glycerol] at 5-10 pmol/ ml (1 OD 260 = 2 pmol) and stored at À80 C.