An OD600 of about 1.0 is ideal, but this is quite flexible. Cultures that are very dense should be avoided because they are likely to be in stationary phase of growth. If the yeast are in stationary phase (not dividing), they become difficult to break and protein is difficult to recover. Often we grow an overnight culture, then pour most of it out in the morning, add another 4-5 mls of media to the same tube and let grow until late afternoon.
If it is important to balance the loads between samples, we take ODs, and then balance the amount of culture to use for extract so that the same total OD600 is used; i.e., use 5 ml of a culture of OD600 0.8, and use 2.5 ml of a culture of OD600 1.6.
Spin yeast down (about 3000 rpm, 3-5 min) and aspirate supernatant.
Re-suspend pellet in 400 µl of 1.5X SDS-PAGE loading buffers containing leupeptin, pepstatin, PMSF. Transfer to an Eppendorf tube containing about 300-400 µl of glass beads on ice.
The inhibitors are really important for protein recovery.
Vortex at top speed at 4°C for 5 minutes.
Use the vortex in the cold room.
To separate the extract from the glass beads, place each tube on top of another Eppendorf and poke a hole in the top and bottom of the tube using an 18-gauge needle. Give the tandem tubes a quick spin in the microfuge. The extract will now be in the bottom tube and the glass beads in the top tube. The extract is ready for heating and loading. Load 20-30 µl/lane.