Superdex 10 300 gl column

PAGE/HPLC-purified RNA and DNA oligonucleotides were reconstituted to 100 μM with nuclease-free distilled water (Invitrogen). RNA:DNA hybrids were formed using 20 μM of each complementary oligonucleotide denatured in 60 mM KCl, 50 mM Tris (pH 8.0) at 95°C for 5 min followed by gradual cooling to room temperature. Resulting RNA:DNA duplexes were concentrated by precipitation with 2.5 volumes of 100% ethanol, 0.3 M sodium acetate (pH 5.2) and resuspended in an appropriate volume of nuclease-free TE (10 mM Tris, 0.1 mM EDTA). Purification was performed on an ÄKTA FPLC™ machine (GE Healthcare) at 4°C by injecting 100 μl volume of nucleic acids onto a 24-ml Superdex 10/300 GL column (GE Healthcare) pre-equilibrated with two column volumes of nuclease-free TE. Elution was performed with equilibration buffer (TE) at a flow rate of 0.4 ml/min, with 100 μl fractions collected into sterile nuclease-free 96-well 2-ml boxes (Greiner Bio-One). Absorbance at 280 nm was recorded for each fraction. Relevant fractions containing the purified hybrid were pooled, quantified and concentrated by ethanol precipitation and analysed by native PAGE.

PDI proteins were reduced with 10 mM DTT for 10 min at room temperature. DTT was removed using a 5 ml of Hitrap desalt column (GE Healthcare) pre-equilibrated with 50 mM Tris/HCl, pH 7.5, buffer containing 150 mM NaCl and 1 mM EDTA (buffer A), according to the manufacturer's protocol. To oxidize the proteins, samples were incubated with 10 mM GSSG for 10 min at room temperature. GSSG was removed by gel filtration using a Superdex 10/300 GL column (GE Healthcare) pre-equilibrated with buffer A. The redox status of all PDI proteins was verified by carrying out AMS alkylation and separation by SDS/PAGE.