Millipore concentrator filters

Recombinant human PKR_1-169 (N-terminal double-stranded RNA binding motifs with residues 1 to 169) as well as full-length PKR were expressed and purified as previously described [14 (link)]. Affinity purified proteins were subjected to SEC using a HiLoad 26/60 Superdex 75 gel filtration column (2.6 x 60 cm, GE Healthcare Life Sciences, USA) for PKR_1-169, or HiLoad 26/60 Superdex 200 size exclusion column for full length PKR (all in 50 mM TRIS (pH 7.50), 100 mM NaCl, and 5 mM 2-mercaptoethanol (Buffer 2)). The elution fractions were monitored by means of absorbance at 280 nm and fractions containing purified protein were combined and concentrated using Millipore concentrator filters (Millipore, USA). Protein purity was confirmed by SDS-PAGE and concentration was determined using the known extinction coefficient as measured by UV-Vis spectrophotometry (NanoDrop2000c, Thermo Scientific, USA).

Affinity-purified protein was subjected to SEC using a Superdex 200 10/300 GL gel filtration column (10 × 300 mm; GE Healthcare Life Sciences, Pittsburgh, PA) in 20 mM HEPES (pH 7.2), 300 mM NaCl, 2 mM dithiothreitol (DTT), 0.1 mM EDTA, and 10% (v/v) glycerol. The eluted fractions were monitored by absorbance at 280 nm. Multiple fractions of individual peaks were combined and concentrated using Millipore concentrator filters (Millipore, Burlington, MA). Protein purity was confirmed by sodium dodecyl sufate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein concentration was determined spectrophotometrically by absorbance at 280 nm using the extinction coefficient (129,830 M⁻¹ cm⁻¹) calculated with the ProtParam tool on ExPASy servers (33 ).