233- Incubate at 4 °C for 12 h ( see Note 13 ).
- Discard supernatant and resuspend the cells in 1 mL of 50 mM
sodium citrate. - Collect the cells via centrifugation at 1,400 × g for 5 min
at 4 °C. - Resuspend the cells in 500 μL of 50 mM sodium citrate con-
taining 0.2 mg/mL RNase A. - Incubate cells at 37 °C for 20–24 h.
- Add SYTOX Green dye to a fi nal concentration of 3.7 μM.
- Incubate at room temperature for 1 h in the dark.
- Collect the cells via centrifugation in microcentrifuge
at 5,600 × g for 2 min. - Discard supernatant and resuspend the cells in 400 μL of
1× PBS. - Collect the cells and resuspend in 400 μL of 1× PBS.
- The cells may be viewed under the microscope or sorted via
fl ow cytometry.
4 Notes
- We buffer our YPD medium to a pH 3.5 using HCl. This
medium is used throughout all experimentation. To prepare
500 mL of acidic YPD medium, dissolve 5 g of yeast extract
and 10 g of Bacto Peptone in 425 mL of water. Add HCl to
the medium until the pH reaches 3.5. The solution will have a
“grainy” appearance. Make up the volume to 450 mL and
autoclave. Make 20 % (w/v) dextrose solution and fi lter-
sterilize. Add 50 mL of the dextrose solution to the autoclaved
solution (2 % fi nal concentration) to make the fi nal acidic YPD
medium. - The constant or crystallizable Fc region of Rabbit IgG binds
with high affi nity to the “protein A” component of the TAP
tag. This antibody can be replaced with other antibodies that
bind protein A, another epitope within the TAP tag or a differ-
ent protein of interest. - When collecting cells or starting any treatment, ensure that the
OD 660 is similar across all samples (OD 660 ± 0.05). - To ensure credibility of heat-stressed samples, limit the time
taken to collect the sample to 10 min. Cells may be collected
at a higher centrifugal force (6,000–8,000 × g ) for less amount
of time (2–3 min) for this time point. - Buffer B is more hypotonic with a lower NP-40 concentration
and is ideal for preparation and analysis of soluble and insoluble
Yca1 in Proteostasis