Grow 100 ml yeast cells in desired media overnight to an A600 of ~1.0 (0.6 to 1.2 works well).
For growth in minimal media, 1 ml of a saturated overnight culture in minimal media (Synthetic dextrose (SD) with only the required amino acids) was inoculated to 100 ml of the same media and grown ~16 hrs at 30 degrees.
Harvest cells and wash with 20 ml of cold extraction buffer in a 50 ml tube.
Resuspend cells in 0.5 ml extraction buffer containing DTT and protease inhibitors in a microcentrifuge tube with a locking top (marsh tube).
Add ~500 microliters of glass beads. In the cold room, shake tubes on the foam ring of the vortex mixer platform at top speed for 1 min. Transfer to ice for 1 min. I have done up to 20 extracts at once.
Repeat for a total of 10 min of vortexing.
Briefly microcentrifuge to remove all liquid and leave behind most of the glass beads.
Centrifuge at top speed at 4 degrees for 15 min and remove supernatant, being careful to avoid any glass beads.
Assay protein concentration using BioRad or Pierce assays. Freeze extracts and store at -80 deg. Expect 10-15 mg/ml protein.
Extract Buffer:
100 mM Tris pH 7.9
250 mM Ammonium Sulfate
1 mM EDTA
10% Glycerol
Before use, add DTT to 0.5 mM (low concentration so IP reactions can be done directly)
And 1X protease inhibitors from the following stock solutions:
0.1 M PMSF (100x) 16 mg/ml Ethanol; Store at -20 degrees
Benzamidine (100X); 31 mg/ml H2O; Store frozen at -20 degrees
Leupeptin (500X); 0.15 mg/ml Ethanol; Store at -70 degrees for less than 6 months
Pepstatin (200X); 0.28 mg/ml methanol; Store at -20 degrees.
Chymostatin (2,500X); 5mg/ml DMSO; Store frozen at -20 degrees
首页
