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Yeast Protein Lysates for SDS-PAGE 1. Collect 1 OD unit of yeast culture. OD unit = ml X OD600 (e.g., 1 OD unit = 1 ml at OD600 1.0).
 

Summary: Yeast Protein Lysates for SDS-PAGE
1. Collect 1 OD unit of yeast culture. OD unit = ml X OD600 (e.g., 1 OD unit = 1 ml at OD600 1.0).
This method may be used without modification for 0.5 to 5 OD units.
2. Microfuge 1 minute at top speed. For larger volumes, centrifuge 5 minutes at 2500 rpm. Wash
with 0.5 ml TE buffer (or dH2O) and microfuge again. Pellets may be stored at -80C.
3. Add 0.5 ml of 0.2 N NaOH + 1% 2-mercaptoethanol to pellet. Vortex to resuspend completely.
NB: Freshly prepare NaOH/2-ME and use at room temperature for optimal lysis. If using frozen
cell pellets, do not thaw pellet prior to addition of NaOH/2-ME (use NaOH/2-ME to thaw pellet).
4. Add 50 l of 50% TCA. Vortex to mix completely. Place on ice 10 minutes (or longer).
5. Spin 2 minutes in microfuge at top speed at 4C.
6. Wash pellet with 0.5 ml 0.1% TCA (or acetone). Hold on ice 5 minutes.
7. Microfuge for 2 minutes at top speed. Washed pellets may be stored at -20C.
8. Add 100 l of freshly made SDS-PAGE sample buffer 2. Vortex vigorously. Disperse with water
bath sonicator for a minute or two. Microfuge a few seconds to collect droplets in bottom of tube.
NB: If more or less than 1 OD of cells was used, add sample buffer at 100 l per 1 OD unit.
9. Boil for 5 minutes. Lock tube cap with plastic closure or use metal rack with locking top.
10. Microfuge for 5 minutes at top speed at 4C. Transfer supernatant to a fresh tube. Do not transfer
pellet. Store at -80C (do not store at -20C).
NB: Try 5 l of sample for a mini gel, or 10 l of sample for a regular gel.
Sample Buffer 2 Stock (SB2)

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine