Calculate concentration with formula below: Decant the PBS wash and aspirate the excess supernatant. Collect the supernatant avoiding the pellet into new microtubes.
If the lysate is still viscous after sonication, passage through a 26 gauge needle to complete fragmentation of nuclear DNA.
Pellet the cells for ten minutes at 2,000 rpm. Do not keep sample in ice bath for an extended period of time.
These tend to aggregate when boiled and the aggregates may not enter the gel efficiently. Spin down membranes by centrifuging the supernatant from Step 6 at 29,000 xg for 45 minutes.
Rinse cells with ice-cold PBS. Slowly and carefully add into media to avoid precipitation.
Pellet cells by centrifugation 1 minute at 1800 x g and remove media. Wash pellet one time with 5 to 10 ml ice cold PBS.
Centrifuge for 30 minutes at 25,000 xg. Please note that in addition to whole cell lysates there are also membrane and nuclear specific preparations that can be used to isolate and concentrate different cellular components.
After immunoprecipitation, kinase activity is measured in one of two ways. Continue Continue. If a large volume of cells is involved, cells may need to be concentrated to a lesser volume of media so that the stimulating reagent is not wasted.
Keep sample at room temperature. Centrifuge nuclei and unbroken cells at 2,900 xg for 20 minutes. This assures that no cells are left behind. Extraction conditions release proteases from the cells, thus the need for protease inhibitors in the lysis buffer.
Output set at 10. Transfer lysate to an appropriate sized tube and boil for an additional 10 minutes. Using a cell scraper or silicone spatula, scrape the cells and transfer the lysate to a 15 ml conical. Treatment Stimulation Note: Rinse the nitrocellulose in 4 to 5 changes of water and continue on with Western blotting protocol.Cell lysis