Preparing yeast protein extracts for IP
1. grow 2 ml overnight culture
2. inoculate 20 ml with the last culture
3. dilute to 200 ml YPD, grow 1.5 hrs
4. spin down, wash 1x50 ml ice cold H2O
5. Lyse in x ml lysis buffer, shake with glass beads in eppendorf tubes 10 min 4C
6. spin down 15 min max rpm, save SNT
7. estimate protein concentration using Biorad/Bradford assay
lysis buffer:
150 mM NaCl
50 mM Tris pH 7.5
10 mM EDTA
10% glycerol
1mM DTT
PPase inhibitors (NaF, pyrophosphate de Na, orthovanadate de Na)
protease inhibitors
2. inoculate 20 ml with the last culture
3. dilute to 200 ml YPD, grow 1.5 hrs
4. spin down, wash 1x50 ml ice cold H2O
5. Lyse in x ml lysis buffer, shake with glass beads in eppendorf tubes 10 min 4C
6. spin down 15 min max rpm, save SNT
7. estimate protein concentration using Biorad/Bradford assay
lysis buffer:
150 mM NaCl
50 mM Tris pH 7.5
10 mM EDTA
10% glycerol
1mM DTT
PPase inhibitors (NaF, pyrophosphate de Na, orthovanadate de Na)
protease inhibitors