HCT116 cells were lysed in Triton X-100 lysis buffer [50 mM tris (pH 8.0), 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM dithiothreitol (DTT), protease inhibitors, and Benzonase]. After centrifugation at 13,000g for 10 min, the supernatants (1 mg of total protein) were collected and incubated with anti-Flag M2 affinity gel at 4°C for 2 hours with rotation. Samples were washed with lysis buffer four times and competed with 3×Flag peptides for 15 min with vigorous agitation. Proteins were resuspended in 5× SDS sample loading buffer, heated to 95°C for 5 min, and subjected to SDS–polyacrylamide gel electrophoresis (PAGE).

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

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.