• 5 ml overnight culture of your favorite yeast in your favorite medium.
• Inoculate 50 ml and grow 30o C shaking O/N until OD600 = 0.8 to 1.2.  For SCD cultures use 1/500 and 1/1500 dilutions.
• Optional: add alpha-factor to 2.5 µM.  Continue shaking at 30o C for 60 min.  
• Add 1 M NaN 3 to 10 mM (final concentration) and move cultures to ice.  Everything must remain cold from here on out.  
• Spin cells 3 K, 10 min at 4o C. 
• Discard supe and resuspend cells with 0.5 ml 10 mM NaN3.  Transfer to 1.5 ml microfuge tube (not autoclaved).
• Spin 3 K, 10 min at 4o C.  Alternatively spin 8 K, 1 min at room temperature.
• Discard supe.  Optional: freeze pellet at -80o C.  
• Resuspend in 1 ml of cold 10 mM NaN3. 
• Measure OD600.  Adjust volumes so that there are an equal number of cells in each sample. A total OD600 of 30 per sample is best. 
• Wash with 1 ml of Lysis Buffer.  
• Spin 3 K 10 min at 4o C and discard supe.  
• Resuspend in 400 ul Lysis Buffer.
• Add a scoop of glass beads to a 0.5 ml PCR tube.  Transfer cell lysate to the PCR tube 
• Vortex 1 min, 4X.  Keep samples cold between vortexing.
• Poke a hole in the bottom of the tube and spin cell lysate in a new microfuge tube 1.5 K or 500 X g, 10 min., 4o C. 
• Transfer liquid from bottom tube into a new microfuge tube.  
• Spin again 1.5 K, 10 min, 4o C and again transfer liquid into a new microfuge tube.
• Add Triton X-100 to 1.5 % and rock for 60 min at 4o C. 
• Spin (3 K, 10 min, 4o C) and transfer the supe to a new microfuge tube.
• Remove 30 ul of liquid and add 30 ul 2X SDS PAGE Sample buffer. This will reflect protein content before Glutathione purification.
• To the remaining liquid, add 100 ul 40 % slurry of Glutathione beads and mix at 4o C for 2 h (overnight is usually fine).  Glutathione beads should be prewashed 3 X with PBS and 1 X with lysis buffer before resuspending as a 40 % slurry in lysis buffer.
• Wash glutathione beads five times with PBS, 1 % Triton X-100, 300 mM NaCl at RT.  Spin 2 K, 5 min, at room temperature.  Rock sample for 5 min between washes.  Change tubes after the first wash to reduce nonspecific binding to the tube itself.
• Resuspend in SDS-PAGE Sample buffer.
• Alternatively elute 3 times with 1-2 column volumes of 5-10 mM reduced glutathione, 50 mM Tris pH 8, and mix with 6X SDS-PAGE sample buffer before stripping the beads with SDS-PAGE sample buffer.  
• Heat to 100o C for 10 min. Then store at -20o C.  Protein is ready to be run on SDS-PAGE Gel.

NOTES

Thanks to Paul DiBello and Jiyoung Cha for their refinements of this protocol.

* Indicates a correction from an eariler version of the protocol. 

2. You can substitute protease inhibitor cocktail (Sigma P8215) for individual protease inhibitors (AEBSF, leupeptin, pepstatin, benzamidine, aprotinin).

3. For lysis to determine phosphorylation, you may wish to add more phosphatase inhibitors (in addition to beta-glycerolphosphate and Na-o-vanadate) to the lysis buffer, or you may wish to omit phosphatase inhibitors altogether:
 

50mM Na-M-Vanadate	200ml	0.5mM
100mM Na-pyrophosphate	2 ml	10mM
2mg/ml Phosvitin	10µl	1µg/ml