To identify in vivo methylation sites of Plk1, 293T cells were transfected with Flag-tagged Plk1. After 36 hours, the cells were harvested and lysated. Flag-Plk1 was immunoprecipitated by α-Flag resin and subjected to SDS-PAGE. The band corresponding to Plk1 was subjected to in-gel trypsin digestion and desalted with C18 tips. The samples were analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) on a Q Exactive-HF mass spectrometer (Thermo Fisher Scientific). The LC-MS/MS data were processed using Proteome Discoverer (Thermo Fisher Scientific) and searched against the Swiss-prot Homo sapiens protein sequence database. The existence of monomethylation was verified on the basis of the occurrence of consecutive y or b ions and the mass increment of 14.016 Da on lysine residues.

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.