Cytoplasmic extracts for IP

Source: Genhong Cheng

Reagents:

Lysis/IP buffer

Volume Stock solution

1.5 ml 5 M NaCl
1 ml 1 M HEPES, PH7.2
0.5 ml 1 M NaF
40 ul 0.5 M EDTA
0.5 ml Triton X-100
Mix well
47 ml H20
Keep on ice, add the follwoing protease inhibitor before use
0.5 ml 100% Aprotonin
5 ul 10 mg/ml Leupeptine
0.5 ml 100 mM PMSF (in isopropanol)

Procedure

• For each IP, take 1-3 x 107 exponentially growing attached cells.
• Before touch cells, chill 1X PBS on ice , make 50 ml lysis/IP Buffer and leave it on ice .
• Suck off medium.
• Add 10 ml 1X PBS (cold) carefully so you do not disrupt the cells.
• Suck off the PBS.
• Add 10 ml 1X PBS (cold) and detach the cells either through pipeting or by scratching.
• Transfer cells to a 15 ml tube (be sure to label the tube).
• Spin down the cells in the bench-top centrifuge at 1000 rpm for 5 minutes at 4 C.
• Suck off supernatant.
• Resuspend cell pellet in 1 ml lysis/IP buffer (cold, be sure add protease inhibitors).
• Transfer cells into a 1.5 ml microcentrifuge tube.
• leave the tube on ice for 30 minute with shaking every 5 minutes.
• Spin cell lysate in a microcentrifuge in the cold room at 2K rpm for 3 minutes
• Transfer the supernatants into a new 1.5 ml microcentrifuge tube.
• The supernatants can be used directly for Western and IP, or can be freezed at -70 C.