Leang, Ching 1-1
Salt Wash Using KCl for Geobacter
(Childers, Spring 2003)
1. For cells grown with Fe(III) oxides, oxalate extract to remove oxides before
collection.
2. Weigh centrifuge tube before collecting cells.
3. Collect cells at 5000 rpm (GSA rotor) for 20 minutes at 4˚C.
4. Wash cells in 30-100 ml 50mM HEPES pH 7.5 plus 1mM MgCl2 and protease
inhibitor.
a. Use Roche Labs Complete Protease Inhibitor.
b. Follow instructions.
5. Collect cells at 7000 rpm (SS34 rotor) for 15 minutes at 4˚C.
6. Weigh pellet to get approximate wet weight of cells
7. Resuspend cells in 50mM HEPES pH 7.5 plus 1mM MgCl2 and 0.5M KCl.
a. Use ~ 1ml per 0.2g wet weight.
8. Put suspension into small serum bottle with a tiny stir bar
9. Stir on ice for at least 20 minutes
a. Put ice and a little water in beaker, enough to cover cell suspension.
10. Remove cells by centrifugation at 7000 rpm (SS34) for 20 minutes at 4˚C.
11. Transfer supernatant to a new tube and keep on ice.
a. This is the 0.5M KCl fraction.
12. Resuspend cell pellet in 50mM HEPES pH 7.5 plus 1mM MgCl2 and 2M KCl.
a. Use ~ 1ml per 0.2g wet weight.
13. Stir on ice for at least 20 minutes
a. Put ice and a little water in beaker, enough to cover cell suspension.
14. Remove cells by centrifugation at 7000 rpm (SS34) for 20 minutes at 4˚C.
15. Transfer supernatant to a new tube and keep on ice.
a. This is the 1M KCl fraction.
16. Store remaining cell pellet at -20˚C for outer membrane fractionation.
17. There is too much KCl in the fractions for PAGE therefore the fractions must
be desalted.
a. Use the Amersham PD-10 columns for desalting as instructed.
b. Use the 50mM HEPES pH 7.5 plus 1mM MgCl2 buffer as above
minus the KCl.
18. Once samples have been desalted, concentrate using 3,000 mw cutoff filter or
10,000 mw cutoff filter.