Yeast Crude Protein Extraction

Materials:

Tabletop centrifuge at 4°C
Ice-cold H2O
Lysis buffer
0.5mm glass beads

Procedure:

1. Grow 5 mL of cells overnight to approximately OD600 = 1.

2. Transfer cells to 15 mL conical tubes and centrifuge at 4°C at 3000 rpm for 5 min.

3. Wash cells with 2 mL ice-cold H2O, centrifuge again, and discard supernatant.

4. Add 50-100uL lysis buffer (10uL for every 1 OD600 unit), resuspend cells, and transfer to an Eppendorf tube.

5. Add an equal volume of 0.5mm glass beads and lyse by vortexing for 5 min at 4°C.

6. Centrifuge at 4°C for 10 min and transfer supernatant to a fresh Eppendorf tube. Store at -70°C.

7. Load 10uL of sample (after adding dye and boiling 3 min).

Lysis Buffer:

20 mM Tris, pH 7.6
100 mM NaCl
1 mM EDTA, pH 8.0
2% Triton X-100
1% SDS

Protease inhibitors

For 1 mL:
20uL 1M Tris
20uL 5M NaCl
2uL 0.5M EDTA
80uL 25% Triton X-100
100uL 10% SDS
10uL 100X Complete protease inhibitors (1 tablet in 500 uL)