From Powers Lab UC Davis
1. Resuspend about 5 OD of cells
(approximately 10 mL of cells at OD600= 0.5) in 0.5 mL of cold TCA buffer which
includes 2X protease inhibitors (and some samples also with 2X phosphatase inhibitors).
2. Prepare tubes with approximately 500 µl of glass beads and 0.5 mL of
cold 30% TCA.
3. Put cells in TCA tubes.
4. Bead-beat in cold 1.5 mL microfuge tubes on high vortex setting. Do this
4X for 30 seconds with cooling on ice during the interim.
5. Do not centrifuge yet. Transfer supernatant to 1.5 mL cold microfuge tubes.
6. Centrifuge 5 minutes in 4°C microfuge.
7. Wash pellet 1X with 1 mL cold (-20°C) acetone. Leave the pellet alone
and invert the acetone about 5-10 times.
8. Remove the acetone completely.
9. Resuspend pellet in 100 µl SDS-PAGE sample buffer. The sample should
be blue. If it is yellow or orange check pH with pH paper and neutralize with
NaOH if necessary. Add 5N NaOH in 0.5µl increments and mix after each
addition until sample is blue.
10. Incubate 5-10 minutes at 65°C.
11. Cool and analyze. Run on 4-15% SDS-PAGE gradient gel from Bio-Rad.
TCA Buffer for 10 mL
200 µl 1M Tris pH 8.0
66.6 µl 7.5 M ammonium acetate NH4OAc
40 µl 0.5 M EDTA (important for protease inactivation)
9.45 mL H2O (or 8.45 ml if adding phosphatase inhibitors)
+ 2X protease inhibitors (see below)
2X Protease Inhibitors
200 µl PMSF stock
20 µl leupeptin stock
20 µl aprotinin stock
2X Phosphatase Inhibitors
500 µl NaF stock
500 µl B-glycerophosphate stock
SDS-PAGE Sample Buffer
900 µl sample buffer
100 µl 1M DTT
10 µl PMSF
1 µl leupeptin
1 µl aprotinin
27 µl NaF (if phosphatase inhibitors are being added)
50 µl B-glycerophosphate (if phosphatase inhibitors are being added)