226
- Vacuum source.
- Scotch Tape.
- YPD medium: 1 % (w/v) yeast extract, 2 % (w/v) Bacto
Peptone, 2 % (v/v) dextrose, pH 3.5. - FM lipophilic styryl dye (e.g., Molecular Probes).
- Phosphate buffered saline (PBS), pH 7.4.
- Aluminum foil.
- 37 % (v/v) Formaldehyde.
- Coverslips: 24 × 50 mm (Fisher Scientifi c).
- Poly- L -Lysine coated glass slides (Sigma).
- Dynabeads (Dynal Biotech).
- Magnetic stand (Dynal Biotech).
- Buffer C: 20 mM HEPES, 0.1 % (v/v) Tween 20, 2 mM
MgCl 2 , 300 mM NaCl, pH 7.4. Store at 4 °C. - Protease Inhibitors (Calbiochem Cocktail Set IV or similar).
- Buffer D:50 mM Tris–HCl, 2 % (w/v) SDS, 0.1 % (w/v) bro-
mophenol blue, 10 % (v/v) glycerol, 150 mM NaCl, pH 6.8. - 0.1 M sodium phosphate buffer: 19 mM monosodium phos-
phate, 81 mM disodium phosphate, pH 7.4. - Ammonium sulfate buffer: 3 M ammonium sulfate, 19 mM
monosodium phosphate, 81 mM disodium phosphate, pH 7.4. - Phosphate buffered saline (PBS), pH 7.4.
- 0.1 M Citric acid, pH 3.1.
- Dimethylformamide (DMF).
- Triton-X 100.
- Antibody: nonspecifi c Rabbit IgG (Chemicon, see Note 2 ).
- 70 % (v/v) Ethanol.
- 50 mM Sodium citrate.
- 0.2 mg/mL RNase A.
- Phosphate buffered saline (PBS), pH 7.4.
- SYTOX Green (Molecular Probes).
3 Methods
- Inoculate a 3 mL aliquot of medium with a single yeast colony.
This will be the starter culture. - Grow for 12–16 h at 30 °C, 225 rpm until OD 660 is at least
between 0.8 and 1.0.
2.5 Vacuole Staining
2.6 Immuno-
precipitation
2.7 DNA Staining
3.1 Normal and Heat
Stress Growth
Conditions
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