Kushnirov (post-alkaline extraction) lysates for western blots:

1. Grow a 10 ml culture

• for repressing conditions, use medium with 5% glucose and grow overnight to OD₆₀₀=0.7-1.5

• to derepress, spin cultures 5' 3K (RC3B centrifuge), discard supernatant in the sink, resuspend cells in 5 mls sterile water, spin again and discard sup, resuspend cells in medium with 0.05% glucose

2. Before starting:

• turn on heat block to 99^o

• thaw 100X protease inhibitors and PMSF

• add protease inhibitors to enough H²O for 2 mls per sample

• add ß-mercaptoethanol to sample buffer, 40µl per 1ml, enough for 500µl per sample

• weigh one Eppendorf per sample and write the weight on the tube

3. Spin cultures in the big centrifuge (RC3Bplus), 5' 3K

4. Dump supernatant into the sink, resuspend cells in 1ml H₂O + protease inhibitors, add ~1µl PMSF

5. Transfer to an Eppendorf tube

6. Spin 5 seconds full speed in the Eppendorf centrifuge

7. Aspirate sup, weigh Eppendorf with cells

8. Resuspend cells in 100µl H2O + protease inhibitors per 0.02 g of cells, add ~1µl PMSF

9. Add an equal volume of 0.2M NaOH, 5' room temperature (do not let these sit longer than 5')

10. Spin for 5 seconds and aspirate sup

11. Add 100µl sample buffer per 0.02g cells (from the weight in step 8), resuspend by pipetting up and down

12. Boil 5'

13. Spin 5', transfer sup to a new tube

14. Run immediately or freeze on dry ice and transfer to -70.

15. To run after freezing: thaw, heat 70^o 10', spin 5'

16. Refreeze as soon as possible

reference: Yeast (2000) 16:857-860