1. Grow 50 ml cells to log phase (OD600 = 0.6-0.8) by diluting 3-5 ml overnight culture in 45 ml YPD and incubating at 30C while shaking (about 2.5 - 4 hrs).
2. Spin down cells, wash in 50 ml ice-cold H2O.
3. Resuspend pellet in 400 µl ice-cold 20% TCA and transfer to a 1.5 mlEppendorf tube.
4. Fragment the cells by adding approximately 150 µl glass beads (Sigma G8772) and vortexing for 15 min at 4C.
5. Transfer the lysate to a fresh Eppendorf tube, wash the glass beads once with 400 µl ice-cold 20% TCA and add this wash fraction to the tube with the lysate. Discard the old tube containing the glass beads.
6. Spin down 6 min at 4C max speed.
7. Remove the supernatant and wash the pellet twice with 800 µl 70% EtOH (resuspending the pellet). Centrifuge 6 min max speed at 4C between the wash steps. Be careful because the protein pellet crumbles easily in EtOH.
8. Remove as much EtOH as possible.
9. Resuspend pellet in 200 µl 1 x Laemmli sample buffer (which will turn yellow due to the remaining TCA in the pellet, which is very acidic).
10. Add unbuffered Tris base (1M) until the lysate turns blue (usually just a few µl).
11. Incubate 5 min at 95C, spin down insoluble matter and transfer the supernatant to a new tube.
12. Load 10-20 µl on gel or store at -20C.
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