Biology Reference
In-Depth Information
Fig.
1
Extraction procedure for phenol and PEG fractionation method
3. Centrifuge at 12,000 ×
g
for 15 min at 4 °C. Transfer phenol
phase to a new 50 mL Nalgene centrifuge tube.
4. Add 4 volume of methanol containing 0.1 M ammonium acetate,
vortex, and place at −20 °C for at least 2 h or overnight to precipi-
tate proteins from the phenol.
5. Wash the protein pellet with 10 mL of 0.1 M ammonium
acetate in methanol. Repeat this step two times.
6. Discard the solution and wash the protein pellet with 10 mL of
80 % acetone. Repeat this step two more times.
7. Semi-dry the tubes in at RT to remove excess liquid.
8. Resuspend protein pellet in rehydration buffer and proceed for
2-DE analysis (IPG gel system).
9. Proceed for 2-DE analysis (Fig.
1
).
