Yeast Protein Lysates for SDS-PAGE
1. Collect 2 ODs unit of yeast culture. Centrifuge for 10 minutes at 2000 rpm.
· 1 OD unit = volume X OD600 (e.g., 2 ODs = 5 mls of culture at OD600 0.4).
· This method may be used for between 0.5 and 5 OD units.
2. Resuspend pellet in 1 ml ice-cold 10% TCA. Transfer to microfuge tube. Place on ice 5 minutes.
3. Microfuge at top speed for 30 seconds. Discard supernatant. Pellet may be stored at -20°C/-80°C.
4. Resuspend pellet in 100 l of 10% TCA. Transfer to 2.0 ml microfuge tube containing 0.25 g acid
washed 0.5 mm glass beads. Place on vortex mixer for 10-30 minutes (depending on speed of
mixer). Monitor with microsope (400X) for cell breakage. Aim for >75% breakage.
5. Transfer lysate to 1.5 ml microfuge tube. Use a gel-loading tip to prevent transfer of glass beads.
Wash glass beads twice with 10% TCA and pool with lysate in 1.5 ml microfuge tube.
6. Microcentrifuge at top speed for 5 minutes at 4°C. Discard supernatant.
7. Wash TCA precipitate pellet with 0.5% TCA or 100% acetone.
8. Resuspend TCA pellet in 100 l of freshly made SDS-PAGE sample buffer 2. Vortex vigorously
and sonify in water bath sonicator to solubilize sample. Spin briefly.
· If more or less than 2 ODs of cells was used, add sample buffer at 50 l per 1 OD unit.
9. Boil for 5 minutes. Lock tube cap with plastic closure or use metal rack with screw top.
10. Microfuge at top speed for 5 minutes at room temperature. Transfer supernatant to a fresh tube.
Do not transfer pellet. Store at -20°C/-80°C.