Protein Extraction from Yeast
Note: always keep tubes on ice.
1) Grow yeast in 10-100mL culture to saturation one or two nights.
2) Pellet cells. 3000rpm, 3min, 4ºC. Dump supernatant.
3) Resuspend in 1ml dH2O. Transfer to tube with purple cap and flat conical bottom.
4) Pellet cells. Dump supernatant.
5) Wash cells twice with 1ml cold PBSMT. Dump supernatant.
6) Add equal amount of glass beads (kept in -20ºC freezer).
7) Add 500_l cold PBSMT, 1ul PLAC, and 1ul PMSF (0.1M).
8) Use bead beater. Shake each tube for 30sec (repeat ~4X).
9) Look at cells under bench scope. Make sure that at least 50% are broken. If necessary, shake again.
10) Centrifuge for 10min at 4ºC. Save the supernatant.
11) Pipette ~400ul of the supernatant into new Eppendorf. Store at -80ºC.
12) Do protein concentration assay (Bradford).
a. Read protein concentration at 595 after vortexing.
b. Blank = 800ul dH2O + 200ul BioRad
c. BSA Samples
i. 1:10 stock = 1mg/ml
ii. 2ul, 4ul, 6ul, 8ul stock + 800ul dH2O + 200ul BioRad
d. Elution Samples
i. 2ul, 5ul sample + 800ul dH2O + 200ul BioRad
e. Use Cricket Graph
i. Enter data.
ii. Graph‡New Graph‡Line y=mg/ml x=OD
iii. Options‡Curve Fit‡Method (linear) .9< r <1
iv. Use formula to calculate concentration
