Download Yeast Whole Cell Extracts

Yeast whole cell extracts
Lenzmeier Research Laboratory
Preparation of yeast whole cell extracts for enzyme kinetics experiments
1. Yeast cells are grown in the appropriate culture medium in a 50 ml volume to late log-phase with an
absorbance around 1.0 at 600nm.
2. The cells are spun down 3 minutes at 4000 rpm in the swinging bucket centrifuge.
3. After centrifugation, the supernatant is discarded and the pellet of cells is kept.
4. The cells are then resuspended in 5 ml of 1x TBS (20 mM Tris-HC1 at pH 7.6, 200 mM NaC1).
5. The cells are the spun down again 3 minutes at 4000 rpm in the swinging bucket centrifuge.
6. After centrifugation, the supernatant is discarded and the pellet of cells is kept.
7. Steps 4-6 are repeated with 1x TBS (for a total of two washes with 1X TBS).
8. Cells are suspended in 1 ml of 1x TBS and then transferred to a microcentrifuge tube.
9. Cells are spun 1 minute at 13,000 rpm in the tabletop centrifuge.
10. The supernatant is discarded and the cells are resuspended in 500 ul lysis buffer (50 mM HEPES-KOH
(or Tris) at pH 7.5,140 mM NaC1, 1 mM EDTA, 10% glycerol, 0.05% NP-40 (or Triton X100), 1 mM
PMSF, 1 mM DTT)
11. Glass beads are added to the cells in lysis buffer until they reach the meniscus of the solution.
12. Cells + lysis buffer + glass beads are then vortexed for 45 minutes in the cold room.
13. The liquid is transferred to new mini centrifuge tube and centrifuged for 10 minutes at 13,000 RPM and
4oC.
14. The liquid is transferred in 100 ul aliquots and either immediately tested in the enzymatic assay or stored
at -20oC for future use.
15. Enzymatic activity levels are determined and normalized to the protein concentration of the extracts.
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