The cytosolic and nuclear fractions were prepared, following a protocol published previously (45) with some modifications. Cells were washed with ice-cold phosphate-buffered saline (PBS) twice and incubated with buffer A [10 mM Hepes·KOH (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.2% NP-40, 1 mM dithiothreitol (DTT), and protease inhibitor cocktail (Roche, Mannheim, Germany)] on ice for 10 min. Samples were then scraped, transferred to microtubes, and homogenized with vigorous shaking. The cytosolic fraction was yielded by collecting the supernatant after two consecutive centrifugations (1500g for 3 min followed by 20,000g for 10 min). The pellet after the first centrifugation was washed with wash buffer [10 mM Hepes·KOH (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and protease inhibitor cocktail] twice, and the resultant pellet was resuspended in buffer B [20 mM Hepes·KOH (pH 7.9), 500 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and protease inhibitor cocktail], incubated on ice for 15 min, and homogenized with vigorous shaking. The nuclear fraction was yielded by collecting the supernatant after centrifugation at 20,000g for 10 min. To verify the nuclear/cytosolic fractioning and to obtain a loading control for each fraction, anti–histone H3 and anti–α-tubulin immunoblottings were conducted (fig. S5, B and C).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.