docs/Protocols/Protein extraction protocol 6-well plate.md

#P

1. If necessary, prepare a protein extraction buffer. See Preparing 1X RIPA with Protease inhibitor and phosphatase inhibitor cocktail
2. Fill a tray with ice. (There is an ice machine in the autoclave room to the left of the cell culture room.)
3. Remove the plate from the incubator and bring it to the lab. (There’s no need to use a laminar flow hood at this point.)
4. Aspirate media.
5. Wash cells with ice cold 1X PBS.
6. Aspirate PBS.
7. Keeping the plate on ice. Add 100 ul of ice cold protein extraction buffer to each well.
8. Leaving the plate on ice, allow cells to sit in the extraction buffer for 10 minutes, agitating the plate every minute or two.
9. Tip the plate towards you so that the protein extraction buffer and suspended cells pool in the bottom corner.
10. If necessary, scrape cells into the extraction buffer with a cell scraper or pipet tip.
11. Using a P200 pipet, transfer the cell suspension to a chilled, prelabeled 1.5-ml tube.
12. Agitate the cells in the ThermoMixer for at 4C/500 rpm for 4 h-overnight.
13. Spin down cell debris at max speed for 25 m at 4C.
14. Avoiding the pellet, transfer 90 ul of supernatant to a fresh, chilled 1.5-ml tube (prelabeled).
15. Transfer remaining 10 ul to 0.2 ml PCR tube strips.
16. Place tubes in a -80C box and note box address.
17. Submit your sample to our inventory/archive by filling out this form: -80C Sample Submission