EM Method for Yeast Cells
1. Prepare 5 OD units of cells in logarithmic growth (e.g., 10 mls of OD600 = 0.5). Spin at 2500
rpm in IEC centrifuge for 10 minutes. The steps below are performed at room temperature.
2. Resuspend (R/S) cell pellet with 1 ml of ddH2O. Transfer to microfuge tube. Spin in microfuge at
5000 rpm for 1 minute. Remove supernatant. Wash cells once again with ddH2O.
3. Wash cells with 50 mM KPi (pH 6.5), 0.5 mM MgCl2.
4. Fixation: R/S cell pellet in 1 ml of 50 mM KPi (pH 6.5), 0.5 mM MgCl2, 2.0% glutaraldehyde.
ImmunoEM: use same buffer with 3% (freshly prepared) formaldehyde + 0.05% glutaraldehyde.
5. Fix for 30 minutes. Use rotator to gently mix cells. Or, place at 4°C overnight.
6. Wash cells with 1 ml ddH2O. Pretreat with 1 ml 0.2 M Tris (pH 9), 20 mM EDTA (pH 8), 100
mM ß-mercaptoethanol for 15 minutes. Wash cells once again with 1 ml ddH2O.
7. Wash cells with phosphate-citrate (PC) buffer: 2.4 g KH2PO4
0.8 g sodium citrate
90 mls ddH2O.
Adjust pH to 5.8 with H3PO4
QS to 100 mls with ddH2O.
8. Digestion: R/S cells in 500 µl PC buffer. Add 50 µl of 10 mg/ml Zymolyase 100T (diluted in
ddH2O; sonify briefly). Place on rotator for 1-2 hours at room temperature. For overnight
fixation, spin down cells, wash with PC buffer, and repeat the digestion step. For ImmunoEM, use 5