Ni nta affinity resin

The cells were further cultured in 1.0 L of LB medium containing 100 µg ml^–1 ampicillin at 37°C until the OD₆₀₀ reached 0.6–0.8. Then, 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was added, and the cells were incubated for 8 h at 30°C. The cells were then harvested by centrifugation and resuspended in lysis buffer containing 20 mM Tris (pH 8.0), 150 mM NaCl, 2 mM β-mercaptoethanol (β-ME), and 10% (v/v) glycerol. Next, the cells were disrupted by sonication for 20 min on ice and centrifuged at 13000 rpm at 4°C for 30 min. The supernatant was then mixed with Ni-NTA affinity resin (GE Healthcare, U.S.A.) and incubated for approximately 30 min on ice. Subsequently, the resulting slurry was loaded onto a column and washed using 200 ml of washing buffer containing 20 mM Tris (pH 8.0), 150 mM NaCl, 2 mM β-ME, 10% (v/v) glycerol, and 30 mM imidazole. The recombinant ComP with the 6 × His tag was eluted with 30 ml washing buffer supplemented with 250 mM imidazole. Then, the eluted fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 15% gel and stained with Coomassie blue. The target protein was concentrated using Centriprep columns (Millipore, U.S.A.) with a buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM β-ME, and 10% (v/v) glycerol.

sNASP was expressed in bacteria as an N-terminal (His)₆ fusion construct using Ni-NTA affinity resin (GE Healthcare) and was further purified by ion-exchange and gel filtration chromatography after cleavage of the (His)₆ fusion by TEV protease, as described previously (25 (link)). sNASP mutants, truncations and the budding yeast homolog Hif1 were expressed and purified using the same method. N-terminal tagged GST-ASF1A was expressed in bacteria and purified over a Glutathione Sepharose resin (GE Healthcare), the GST tagged removed with Precision Protease cleavage, and further purified using anion exchange and gel filtration chromatography. Mb13 was expressed as a (His)₆, Avitag fusion in bacteria, and after cleavage of the (His)₆ fusion, was further purified by ion exchange and size exclusion chromatography. Full length Xenopus histones H3 and H4 were purified and refolded as described previously to form the H3–H4 dimer/tetramer (33 (link)). For reconstitution of monomeric histones with chaperones, H3 and H4 were dialyzed to water and then added directly to the chaperone. MBP–H3 (116–135) and MBP-mb1 were expressed in the same way as sNASP and purified over Dextrin Sepharose (GE Healthcare) in 20 mM HEPES–KOH pH 7.5 and 0.5 M sodium chloride, eluted in the same buffer supplemented with 10 mM maltose. Maltose was removed by dialysis prior to storage at –80°C.