获取chromosome-associated protein的方法

阿平

Mitotic chromosomes were solubilized at 4°C with Empigen lysis buffer [50 mM Tris-HCl, pH 9.0, 25 mM KCl, 4 mM EDTA-KOH, 10% glycerol, protease inhibitors, 1 mM Na3VO4 and 1% Empigen BB (Calbiochem)], as described for nuclear matrices (Staufenbiel and Deppert, 1984). (3)
Mitotic chromosomes were treated with 80 µg/ml DNase I in DNasebuffer (20 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 3 mM CaCl2 and protease inhibitors), as described (Gasser and Laemmli, 1987; Taagepera et al., 1995). The supernatants (S-1) were separated and the pellets were incubated for 20 minutes at 4°C in histone buffer (10 mM Tris-HCl, pH 9.0, 2 M NaCl, 10 mM EDTA, 0.1% CHAPS and protease inhibitors). After the supernatants (S-2) were collected, the residual pellets were recovered as scaffolds. The S-1 and S-2 fractions were combined as nonscaffolds.

参考文献：Overexpression of the Csk homologous kinase (Chk tyrosine kinase) induces multinucleation: a possible role for chromosome-associated Chk in chromosome dynamics.,    Naoto Yamaguchi1 et al,(JSC,2001)