For [35S]-methionine incorporation, cells were incubated with radiolabeled [35S]-methionine for 30 min at normal cell culture conditions. Cells were washed with PBS and lysed with passive lysis buffer (Promega). For [3]H-uridine incorporation, cells were incubated with radiolabeled [3]H-uridine for 30 min at normal growth conditions. Cells were washed with PBS and lysed with whole-cell lysis buffer. Protein was precipitated using trichloroacetic acid at a final concentration of 25%, and protein was captured on glass fiber filter paper (GE Healthcare). Captured protein was washed with 70% industrial methylated sprit (IMS) and acetone before the addition of 2 ml of Ecoscint scintillation cocktail (National Diagnostics), and radioisotope incorporation was quantified using a Wallac WinSpectral 1414 liquid scintillation counter. Each sample was carried out in triplicate, and spectral counts per minute were normalized to the total amount of protein for each sample.

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.