Preparationofcytoplasmicextractsfortheapplicationinacellfreesystem

Characteristics of this procedure:
Cells are grown to 80% confluency, then harvested, washed and disrupted in KPM buffer by freezing-thawing cycles with liquid nitrogen essentially as described by Fearnhead et al., 1997, Genes Dev. 11: 1266-76.

1. Grow cells in a 160 cm² flask (25 ml medium) up to about 80% cell density.

2. Remove the RPMI medium and detach cells by incubating with 10 ml trypsin/EDTA (Sigma) for 5 min at 37°C

3. For the inactivation of trypsin, add 10 ml of Bovine Calf Serum (BCS) to the trypsinized detached cells. Combine harvested cells with RPMI which was previously removed and spin at 200xg for 5 min.

4. Wash cells once with 5 ml of fresh RPMI medium.

5. Wash cells once with 5 ml icecold PBS.

6. Wash cells once with 1 ml icecold KPM (without Cytochalasin B).

7. Resuspend cells in about 1 volume (same volume as cell pellet) of KPM (containing 10 µg/ml Cytochalasin B).

8. Lyse the cells by freezing the cell-suspension in liquid nitrogen and successively thawing them in a warm water bath. Repeat three times.

9. Spin lysate at 16000 g (14000 rpm in Eppendorf microfuge) for 20 min at 4°C

10. The supernatant is the cytoplasmic extract that can be used in cell-free experiments. If necessary, the supernatant can be spun a second time to remove residual membrane fragments.

11. Measure protein content (will be approximately 15 µg/µl) and store extracts at -70°C in aliquots until required (up to several weeks).

MATERIAL

KPM BUFFER also called Extract Preparation Buffer (EPB):

COMPOSITION:

RECIPE for 5 ml:

50 mM PIPES (pH 7.0)
1 mM EGTA
50 mM KCl
2 mM MgCl2
1 mM DTT
0.1 mM PMSF
2 µg/ml Leupeptin
2 µg/ml Pepstatin
2 µg/ml Aprotinin
with or without 10 µg/ml Cytochalasin B

2.5 ml of 100 mM PIPES, pH 7.0 incl.
2 mM EGTA
250 µl of 1 M KCl
100 µl of 100 mM MgCl
5 µl of 1 M DTT
50 µl of 10 mM PMSF
10 µl of 1 mg/ml Leupeptin
10 µl of 1 mg/ml Pepstatin
10 µl of 1 mg/ml Aprotinin
2.04 ml H₂O nuclease free