Hiprep 26 60 sephacryl s 500 hr column

To purify PPV VLPs by IEX chromatography, yeast cells were suspended in PBS buffer pH7.4 and disrupted by high-pressure homogenization. Cell lysates were subsequently adjusted to pH 4.0. After centrifugation at 10,000 rpm, 4 °C for 30 min, the supernatants of pH adjusted cell lysate were loaded onto an XK 50/30 column (GE Healthcare) packed with 400 ml of Capto S ImpAct resin. Binding VLPs were eluted with 20mM sodium acetate buffer containing 500 mM NaCl. To elevate the recovery of VLPs, the precipitates of pH adjusted cell lysates were redissolved in an equal volume of 20mM Tris-HCl buffer pH 8.0. Through centrifugation, the clari ed supernatant were loaded onto an XK 50/30 column packed with 400 ml of Capto Q XP resins. After elution with 20mM Tris-HCl buffer pH 8.0 plus 500 mM NaCl, fractions were dia ltrated for 10 volumes of PBS on ÄKTA ux (GE Healthcare, USA) equipped with a 750 kDa column (11-0005-50, GE healthcare). Further polishing puri cation of PPV VLPs was performed on an AKTA Puri er 100 (GE Healthcare, USA) using a HiPrep™ 26/60 Sephacryl® S-500 HR column (GE Healthcare). About 4ml IEX puri ed sample was injected and eluted with PBS at a rate of 0.5ml/min. Protein concentration was measured by the BCA Protein Assay Kit (23250, Thermo Fisher Scienti c).