Superdex 200 pc 3.2 30 column

Trypsin was added in a 1:200 weight ratio to a solution of the purified GAN–GINS full-length complex, and the mixture was incubated at room temperature. Aliquots were removed at various time intervals and analyzed by gel filtration chromatography using a SMART system and a Superdex 200 PC 3.2/30 column (GE Healthcare) (Figure 2A). Molecular mass markers used were thyroglobulin (670 K), immunoglobulin (158 K), ovalbumin (44 K) and myoglobin (17 K) (Bio-Rad). Aliquots of the fractions were subjected to 5–20% gradient SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining. For crystallization, the digestion reaction was stopped at 10 min by adding an excess amount of PMSF, and then the truncated GAN–Gins51C complex was purified by anion exchange column chromatography. The Hitrap Q column (5 ml) was equilibrated with buffer D (50 mM Tris-HCl, pH 8.0, 0.5 mM DTT, 0.1 mM EDTA, and 10% (v/v) glycerol). The trypsin-digested complex was loaded onto the column, which was developed with a linear gradient of 0.2–0.8 M NaCl in buffer D. Fractions containing the GAN–Gins51C complex were pooled and concentrated to about 10 mg/ml, using an Amicon Ultra 4 centrifugal concentrator (molecular-weight cut-off 3000, Millipore).

Purified proteins and complexes were pre-incubated at 30°C for 30 min with agitation and subsequently loaded on a Superdex 200 PC 3.2/30 column (GE Healthcare) equilibrated with 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM DTT, 1 mM MgCl₂ and 5% (v/v) glycerol. For the experiments described in Figure 1, UPF2 (5.7 μg) was either incubated with UPF3b (6 μg), eRF1 (5 μg) (1:2.65 UPF2/UPF3b molar ratio) or eRF3a (4 μg) (1:1.5 UPF2/eRF3a molar ratio), in 50 μl reaction and the mixtures were fractionated by size exclusion chromatography (SEC). Fractions containing the proteins were analyzed by SDS-PAGE and Oriole Fluorescent Gel Stain (Bio-Rad).

αTAT1 buffer was exchanged into BRB80 with 1 mM DTT and KCl at either 10 or 200 mM using Zeba spin desalting columns (89882; Thermo Scientific, Rockford, IL). A Superdex 200 PC 3.2/30 column (17-1089-01; GE Healthcare) equilibrated in the appropriate buffer was loaded with 50 μl of αTAT1 and run at 4°C with a flow rate of 50 μl/min.