Hiload 16 600 superdex s75 pg column

As described previously46 (link), expression and purification of the heterotrimeric Asf1(1–169)/H3(60–135)/H4(20–102) complex from E. coli yields a large excess of Asf1 not in complex with H3/H4 that can be separated from the Asf1/H3/H4 complex on a HiLoad 16/600 Superdex S75 pg column (GE Healthcare). We collected this excess Asf1 to be used for complex formation with either FL H3.3/H4 or FL H3.1/H4. Complexes were formed through mixing a twofold excess of Asf1 with H3.3/H4 or H3.1/H4 in a buffer containing 20 mM HEPES pH 7.5, 2.0 M NaCl and 1 mM TCEP, this mixture was then dialyzed overnight into a buffer containing 20 mM HEPES pH 7.5, 750 mM NaCl and 1 mM TCEP. The mixture was then injected onto a HiLoad 16/600 Superdex S75 pg column (GE Healthcare) to separate the Asf1/H3/H4 complex from the excess Asf1. The resulting Asf1/H3/H4 was used for ITC studies.