Yeast chromatin fractionation

Yeast chromatin fractionation

 Yeast chromatin fractionation was carried out as previously described (Keogh et al., 2006), with slight modification. 50 OD yeast cells were collected, then washed successively with 10 mL distilled water and 10 mL SB (1 M Sorbitol, 20 mM Tris-Cl pH 7.5). Washed pellets were stored at -80°C. Thawed cell pellets were successively washed with 1 mL PSB (20 mM Tris-Cl pH 7.5, 2 mM EDTA, 100 mM NaCl, 10 mM β-Mercaptoethanol) and SB, then resuspended with 750 uL SB. Yeast cell walls were digested by the addition of 100 uL Zymolase (10 mg/mL, Seikagaku) for 1 hour at RT. After the digestion, 750 uL ice-cold SB was added, then spheroplasts were collected by gentle centrifugation (2K, 5 min ) and washed once with 1 mL ice-cold SB. The spheroplasts were resuspended by 500 uL EBX (20 mM Tris-Cl pH 7.5, 100 mM NaCl, 0.25 % Triton X-100, 15 mM β-Mercaptoethanol), and then 1.25uL 100 % Triton X-100 was added to lyse outer cell membrane. Cells were placed on ice for 10 min with occasional mixing. About one-tenth volume of the cells was taken and aliquoted as ‘Total’. 1 mL NIB (20 mM Tris-Cl pH 8.0, 100 mM NaCl, 1.2 M Sucrose, 15 mM β-Mercaptoethanol ) was layered over the remainder, then centrifuged for 15 min at 12K, 4°C. The upper layer was taken as cytoplasmic fraction. Glassy, white nuclear pellets were resuspended by 500 uL EBX, then 3.75 uL 100 % Triton X-100 was added to the resuspended nuclear pellets. The resuspended nuclear pellets were incubated on ice for 10 minutes with gentle mixing every few minutes. Chromatin was pelleted by centrifugation (15K, 10 min, 4°C). The supernatant was stored as ‘Nucleoplasm’. The chromatin fraction was resuspended by sample buffer then boiled for Western blot experiments.

Reference
Keogh et al (2006) Nature 439:497-501, PMID: 16299494

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