231- Discard supernatant and resuspend the beads in 1 mL of PBS
containing 1 % (v/v) Triton-X 100 and wash for 10 min on
nutator. - Discard supernatant and wash the beads twice with 1 mL PBS
for a 1 min. - Discard supernatant and perform four washes, 10 min each
with 1 mL of 0.1 M citric acid. - Discard supernatant and repeat step 13.
- Resuspend the antibody-conjugated beads in 400 μL of PBS.
- Store beads at 4 °C. The antibody-conjugated beads should be
used within 14 days of preparation. - Grow cells to OD 660 between 0.6–0.8 and collect as described
in Subheading 3.1. - Resuspend the cells in 300 μL of buffer C containing 0.5 %
(v/v) protease inhibitors. - Lyse the cells and elute as described in Subheading 3.2 ,
steps 4 – 10. - Centrifuge lysate at 20,000 × g for 15 min at 4 °C.
- Collect supernatant and transfer to a fresh microcentrifuge tube.
- Determine protein concentration.
- Aliquot 15 mg of protein lysate.
- Add 50 μL of conjugated magnetic beads to lysate.
- Mix sample via rotation at 4 °C for 3 h.
- Place sample on magnetic stand and wash the beads with 1 mL
of cold buffer C for a total of fi ve times. - After fi nal wash, discard the residual liquid.
- Resuspend the beads in 40 μL of buffer D.
- Elute co-purifi ed protein by heating the mixture at 65 °C for
10 min. - Place tube on the magnet to separate the beads.
- Transfer supernatant to a new microcentrifuge tube.
- Add 2-β-mercaptoethanol to a fi nal concentration of 100 mM.
- Boil sample at 100 °C for 5 min and analyze via SDS- PAGE
and silver staining. - Grow cells in 5 mL of YPD medium to an OD 660 between
0.4 and 0.5. - Collect the cells by centrifugation at 4,600 × g for 5 min at
room temperature. - Discard supernatant and resuspend the cells in 2 mL of YPD.
3.6 Vacuolar
Staining
Yca1 in Proteostasis