Model 491 prep cell apparatus

The nucleosomes were reconstituted by the salt dialysis method, as previously described (32 (link)). The DNA fragments for the nucleosome reconstitutions were amplified by PCR and purified by non-denaturing PAGE, using a Model 491 Prep Cell apparatus (Bio-Rad). Each DNA fragment was mixed with the purified histone octamer, and then dialyzed against reconstitution buffer-high, containing 10 mm Tris-HCl (pH 7.5), 2 m KCl, 1 mm EDTA (pH 8.0), and 1 mm DTT. Nucleosomes were reconstituted by decreasing the KCl concentration to that in reconstitution buffer-low, containing 10 mm Tris-HCl (pH 7.5), 0.25 m KCl, 1 mm EDTA (pH 8.0), and 1 mm DTT. The nucleosomes were then purified by non-denaturing polyacrylamide gel electrophoresis using a Model 491 Prep Cell apparatus (Bio-Rad), and stored at −80°C in nucleosome-buffer, containing 20 mm Tris-HCl (pH 7.5), 1 mm DTT, and 5% glycerol.

The NCPs were prepared by the salt dialysis method with the palindromic 146 base-pair α-satellite DNA fragment, as described previously^{1 (link),65 (link)}. The DNA fragment containing one half of the α-satellite DNA fragment in pGEM-T Easy vector was amplified in the E. coli strain DH5α, and was excised from the plasmid DNA by EcoR V (Takara). The DNA fragment was then dephosphorylated by alkaline phosphatase (Takara), and was further cleaved by EcoR I. The DNA fragment was purified by DEAE-5PW anion-exchange column chromatography (TOSOH). The DNA fragment was self-ligated by T4 DNA ligase (NIPPON GENE), and the resulting DNA fragment was further purified by DEAE-5PW anion-exchange column chromatography (TOSOH).
The reconstituted NCPs were purified by non-denaturing gel electrophoresis, using a Prep Cell model 491 apparatus (Bio-Rad). For the H2A/H2A.B heterotypic NCP preparation, the 146 base-pair DNA, the H2A-H2B dimer, the H2A.B-H2B dimer, and the H3.1-H4 tetramer were mixed in a 1:0.85:2.55:1.7 molar ratio. The NCPs were reconstituted by the salt dialysis method. As a result, H2A/H2A, H2A/H2A.B, and H2A.B/H2A.B NCPs were reconstituted. The resulting three types of NCPs were separated by non-denaturing gel electrophoresis, using a Prep Cell model 491 apparatus (Bio-Rad), and the heterotypic NCP was selectively purified.