Samples were sonicated three times on ice using a high-intensity ultrasonic processor (Scientz) in lysis buffer (8 M urea, 1% protease inhibitor cocktail, 3 μM TSA, and 50 mM NAM). The remaining debris was removed by centrifugation at 12,000g at 4°C for 10 min. The supernatant was then collected, and protein concentration was determined with a BCA kit according to the manufacturer’s instructions. For digestion, the protein solution was reduced with 5 mM dithiothreitol (DTT) for 30 min at 56°C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. The protein sample was then diluted by adding 100 mM triethylammonium bicarbonate (TEAB) to urea concentration of less than 2 M. Last, trypsin was added at 1:50 trypsin-to-protein mass ratio for the first overnight digestion and 1:100 trypsin-to-protein mass ratio for the second 4-hour digestion.

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.