Jupiter 5 μm c18 300 preparative column

SPOP-MATH (residues 28 to 164) WT and S119D, S119A and S199N mutants and 53BP1 (residues 1606 to 1656) were cloned in a pET-based expression vector with an N-terminal His₆ tag cleavable by PreScission protease. Two extra residues, Gly and Trp, were included at the C terminus of 53BP1 for protein quantification by ultraviolet light absorbance. The five proteins were expressed in BL21 (DE3) cells grown in appropriate media (LB media, ¹⁵N-enriched M9 media, or ¹³C-,¹⁵N-enriched 9 media) at 37°C to OD₆₀₀ (optical density at 600 nm) 0.6 and then at 15°C for 16 hours following induction with isopropyl-β-d-thiogalactoside (final concentration, 0.5 mM). The harvested cells were lysed with a microfluidizer (Avestin Emulsiflex C5). The proteins were purified by Ni²⁺–nitrilotriacetic acid agarose chelation chromatography (QIAGEN) and incubated with PreScission protease overnight at 4°C to cleave the His₆ tag. The proteins were further purified by size exclusion chromatography using a HiLoad 16/60 Superdex 75 column (GE Healthcare). The 53BP1 SBM synthetic peptide (residues 1636 to 1650) was purified by reversed-phase chromatography using a Jupiter 5-μm C18 300 preparative column (Phenomenex).