Cells were lysed in lysis buffer [0.5% Triton X-100, 20 mM Hepes (pH 7.4), 150 mM NaCl, 12.5 mM β-glycerolphosphate, 1.5 mM MgCl2, 2 mM EGTA, 10 mM NaF, 1 mM Na3VO4, and 2 mM dithiothreitol (DTT)] containing protease inhibitors. Lysates were centrifuged and incubated with indicated antibodies at 4°C overnight. The next day, prewashed protein A/G beads (Pierce) were added and incubated at 4°C for 4 hours. The beads were washed with cold phosphate-buffered saline (PBS) four times and eluted with DTT-containing SDS sample buffer by boiling for 10 min before immunoblotting.

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.