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Analytical glycerol-gradient centrifugation analyses of Rif1 protein

HM Hisao Masai
RF Rino Fukatsu
NK Naoko Kakusho
YK Yutaka Kanoh
KM Kenji Moriyama
YM Yue Ma
KI Keisuke Iida
KN Kazuo Nagasawa
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In 2.2-ml centrifuge tubes, 15–36% glycerol-gradient was made in 20 mM Tris-HCl (pH7.8), 150 mM KCl, 1 mM EDTA, 0.5 mM DTT and cOmplete™ Protease Inhibitor Cocktail (Roche). Twenty µl of a peak fraction of the Ni2+-NTA column (0.22 µm-filtrated) was diluted 5-fold, and layered on top of the gradient. One hundred µl of molecular size standards was similarly layered on top of another tube. These tubes were centrifuged at 40,000 rpm for 16 hr in Beckman TLS55 rotor at 4 °C. 0.1 ml-fractions were removed from top to bottom, and subjected to SDS-PAGE followed by anti-DDDDK blotting. The sedimentation pattern of molecular size standards was analyzed by SDS-PAGE followed by silver-staining.

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