Protein samples were prepared as described in the ChIP assay. Cells were grown in 100 ml of YPD to OD600 = 1.0. Cells were harvested and washed twice with precooled PBS. Harvested cells were divided into two microfuge tubes and resuspended in 800 μl of lysis buffer with protease inhibitors and phosphatase inhibitors [150 mM NaCl, 50 mM tris-Cl (pH 7.5), 0.15% NP-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, leupeptin (1 μg ml−1), pepstatin (1 μg ml−1), 10 mM sodium fluoride, 20 mM β-glycerolphosphate, 10 mM sodium orthovanadate, and 10 mM sodium pyrophosphate] for each tube. Cells were disrupted by bead beating using 0.5-mm glass beads. After clearance of cell debris by centrifugation at 14,000 RPM, 1.5 ml of aliquot was incubated with 30 μl of IgG-Sepharose beads (GE Healthcare) for Rad52-TAP. After incubation at 4°C for 4 hours, the beads in the microfuge tube were collected by centrifugation at 2000 RPM. The beads were washed five times with lysis buffer. Washed beads were mixed with 30 μl of 2× sample buffer and boiled for 5 min at 95°C. SDS-PAGE was performed with 8% separating gel. Precipitated proteins were visualized by Coomassie blue staining. Mass spectrometry analysis was performed by the Proteomics Core Facility at the School of Biological Sciences in Seoul National University, which is supported by the Center for RNA Research, Institute for Basic Science.

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.