Leang, Ching 1-1
Preparation of Outer Membrane Fraction (Geobacter)
Susan Childers 2003
Harvest cells by centrifugation at 5000 rpm for 20 minutes at 4oC.
Wash in 30 ml PBS*
Pellet cells at 8000 rpm for 10 minutes at 4oC.
Resuspend in 3-5ml 50mM Tris, pH 7.4.
Disrupt cell by French press at 10,000psi
Repeat press a second time
Remove debris by centrifugation at 8000 rpm for 14 minutes at 4oC.
Remove supernatant to ultracentrifuge
a. Keep a small amount (0.2-0.5ml) for the CRUDE sample
9. Pellet cell membranes by ultracentrifugation at 50,000 rpm for 1 hour at 4oC.
10. Remove supernatant and store as SOLUBLE (CYTOPLASMIC) fraction.
11. Resuspend pellet in 50mM Tris, pH 7.4 (~2ml).
12. Add lauryl sarkosine to 1% final concentration from a 10% stock
a. Use Calbiochem lauroyl sarcosine, Na salt to make 10% stock.
13. Add tiny stir bar to tube
14. Mix in an ice water bath on a stirplate for 0.5-1hour.
15. Spin at 50,000 rpm for 45 minutes at 4oC.
16. Remove supernatant and store as INNER MEMBRANE 1.
17. Add lauryl sarkosine to 1% final concentration from a 10% stock
a. Use Calbiochem lauroyl sarcosine, Na salt to make 10% stock.
18. Mix in an ice water bath on a stirplate for 0.5-1hour.
19. Spin at 50,000 rpm for 45 minutes at 4oC.
20. Remove supernatant and store as INNER MEMBRANE 2.
21. Resuspend pellet in 50mM Tris, pH 7.4 and 0.1% SDS
22. Use about 0.1-0.2ml
23. This is the outer membrane (OM) fraction.
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*PBS (phosphate-buffered saline):
NaCl
KCl
Na2HPO
KH2PO4
8g/L
0.2g/L
1.44g/L
0.24g/L
***Can also use 50 mM HEPES, 1mM MgCl2 (pH 7.5) for buffer.