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Yeast small scale whole cell extract
Small Scale Yeast Whole Cell Extract for IP
Steve Hahn
August 2007

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
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

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