Summary: Auble Lab
Make Whole Cell Extract:
1. Grow 100ml of cells in YPD at 30 C to OD600 of ~1.0. Add 1.35 ml 37%
formaldehyde and incubate cells at 30 C for fifteen minutes. (For heat shock, add
100ml pre-warmed 42C YPD to culture and transfer cells to 35C water bath for 45
minutes. After 30 minutes, add 2.7 ml 37% formaldehyde per 100ml of media
and continue incubation for 15 minutes more.)
2. Spin cells down in SLA-1500 at 6K for 5', discard supernatent, and wash with
80ml cold TBS (30ml 5M NaCl, 20ml 1M Tris pH 7.5/per 500ml).
3. Repeat wash twice and resuspend pellet in ~2ml cold TBS. Divide volume into 3
fast prep tubes. Spin down pellet and aspirate TBS. Freeze at 80.
4. Suspend each pellet in ~600ul of FA-lysis buffer with 140mM NaCl with fresh
protease inhibitors added.
5. Break cells with glass beads in fast prep machine for 45 seconds (7 times) with a
minute in between each.
6. Puncture tube with needle and spin down in small glass tube in clinical centrifuge
at 2K for 3'.
7. Resuspend everything in tube and transfer to Eppendorf.
8. Sonicate each tube at 30% output, 90% duty cycles for 5 pulses. Alternate putting