Mono q pc 1.6 5 column

HeLa S3 nuclei from 2 × 10⁹ cells were prepared by hypotonic lysis and extracted with 400 mM ammonium sulfate. Dialyzed nuclear extracts (250 mM KCl) were centrifuged for 10 min at 23,500 × g at 4 °C. Supernatants were incubated with 300 µl of packed anti-FLAG M2 beads (Sigma) for 4 h at 4 °C. After incubation, beads were transferred into a gravity flow column and washed with 10 volumes of wash buffer (20 mM HEPES-KOH pH 7.9, 200 mM KCl, 1 mM EDTA, 10% glycerol, 0.01% NP-40, 1 mM 2-mercaptoethanol, 0.2 mM PMSF) by gravity flow. FLAG-DAXX-containing complexes were eluted in 250 µl fractions of elution buffer (wash buffer supplemented with 0.3 mg ml 3×FLAG peptide). Elutions fractions containing FLAG-DAXX were pooled and adjusted to 150 mM KCl by addition of buffer A (20 mM HEPES-KOH pH 7.9, 1 mM EDTA, 0.01% NP-40, 10% glycerol). Pooled fractions were loaded onto a Mono Q PC 1.6/5 column (GE Healthcare; column volume: 0.101 ml) and separated by a gradient from 15% buffer B (20 mM HEPES-KOH pH 7.9, 1 M KCl, 1 mM EDTA, 0.01% NP-40, 10% glycerol) to 100% buffer B (gradient length: 20 column volumes). Fractions of 150 µl were collected.

The following procedures were performed at 4°C using the FPLC and SMART systems (GE Healthcare). Soluble materials from Escherichia coli BL21 (DE3) cells were dissolved in buffer A (20 mM sodium phosphate (pH 7.2), 0.3 M NaCl, 10% glycerol, and 10 mM β-mercaptoethanol), passed through Hitrap DEAE (GE Healthcare), and then loaded onto TALON resin (Clontech). After sequential washes with buffer A and buffer B (20 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 10% glycerol, and 10 mM β-mercaptoethanol), the bound materials were eluted with 0.2 M imidazole in buffer B and loaded onto anti-FLAG M2 agarose. After a wash with buffer B, the bound materials were eluted with buffer B containing 0.1 mg/ml FLAG peptide (Sigma) and then loaded onto anti-HA-agarose (Sigma). After a further wash with buffer B, the bound materials were eluted with buffer B containing 0.1 mg/ml HA peptide (Sigma). The PCNA-enriched fractions detected by SDS-PAGE and Coomassie Brilliant Blue staining were loaded onto a MonoQ/PC1.6/5 column (GE Healthcare) and the proteins were eluted with a linear gradient of NaCl (0.1–0.5 M) in 20 mM sodium phosphate (pH 7.2), 0.1 mM EDTA, 10% glycerol, and 10 mM β-mercaptoethanol.