227- Transfer between 5 and 10 μL of culture to fresh 50 mL of
media. - Incubate freshly inoculated 50 mL culture at 30 °C, 225 rpm.
Incubation time depends on doubling time of yeast strain used. - At OD 660 0.6–0.8, collect cells via centrifugation at 4,600 × g
for 5 min at room temperature ( see Note 3 ). - Discard supernatant and resuspend the cells in sterile auto-
claved water. - Centrifuge at 4,600 × g for 5 min at room temperature to col-
lect the cells. - Discard supernatant and store cell pellet at −80 °C. Cells may
be stored up to 3 months at −80 °C. - For heat stress and recovery, prepare four additional fl asks with
50 mL media and inoculate as stated in steps 3 and 4. Use the
same starter culture for all samples. - At OD 660 0.6–0.8, transfer culture to 42 °C and incubate for
1 h, 225 rpm. - Collect heat-stressed cells as described in steps 5 – 8 ( see Note 4 ).
- For recovery, after 42 °C treatment, transfer cultures to 30 °C
and incubate further at 225 rpm. - Collect cells as described in steps 5 – 8 every 30 min.
- Thaw frozen cells on ice.
- Add 300 μL of ice-cold buffer A or buffer B supplemented
with 0.5 % (v/v) protease inhibitors ( see Note 5 ). - Transfer cell suspension to a chilled microcentrifuge contain-
ing 0.7 g of acid-washed glass beads. - Lyse cells by placing the microcentrifuge tube containing the
cell suspension onto the disruptor vortex for 1 min at 4 °C. - After vortex, let suspension stand at 4 °C for 1 min.
- Repeat steps 4 and 5 for fi ve more cycles.
- After lysis, use needle to pierce the bottom of the microcentri-
fuge tube. - Remove needle and place into chilled 15 cm culture tube on
ice ( see Note 6 ). - Elute mixture from microcentrifuge tube via centrifugation at
260 × g for 1 min at 4 °C. - Transfer entire extract from culture tube to fresh microcentri-
fuge tube on ice. - Clear cell debris via centrifugation at 350 × g for 1 min
at 4 °C. - Transfer supernatant to a new microcentrifuge tube on ice.
3.2 Protein
Extraction
Yca1 in Proteostasis