Biology Reference
In-Depth Information
11. Balance the centrifugation tubes (the difference should be
lower than 5 mg) and spin at 24,000 ×
g
for 1 h in an ultracen-
trifuge equipped with a swinging bucket rotor.
12. Collect the OB fl oating fraction with a spatula (
see
Note 12
).
13. Resuspend thoroughly the collected OB fraction in 2 mL of
IB5 (
see
Note 11
).
14. Take away 30
L of resuspended OBs to control the OB isola-
tion. Make the OB suspension up to 30 mL with IB5.
15. Distribute the suspension into six centrifugation tubes and
overlay with 5 mL of IB6.
16. Balance the centrifugation tubes (the difference should be
lower than 5 mg) and spin at 24,000 ×
g
for 1 h in an ultracen-
trifuge equipped with a swinging bucket rotor.
17. Collect the OB fl oating fraction with a spatula.
18. Resuspend thoroughly the collected OB fraction in 2 mL of
IB1 (
see
Note 11
). Take away 30
μ
L of resuspended OBs to
control the OB isolation. At this moment, the isolation can be
interrupted until the next day and the OB suspension stored
overnight at 4 °C.
19. Add 18 mL of 9 M urea to 2 mL of OB suspension, slightly
mix, and agitate at room temperature for 10 min.
20. Distribute the suspension into four centrifugation tubes and
overlay with 5 mL of IB4.
21. Balance the centrifugation tubes (the difference should be
lower than 5 mg) and spin at 24,000 ×
g
for 1 h in an ultracen-
trifuge equipped with a swinging bucket rotor.
22. Collect the OB fl oating fraction with a spatula (
see
Note 12
).
23. Resuspend thoroughly the collected OB fraction in 2 mL of IB1.
μ
24. Take away 30
L of resuspended OBs to control the OB isola-
tion and make the OB suspension up to 15 mL with IB1.
25. Add 15 mL of hexane, slightly mix, and agitate at room tem-
perature for 10 min to test OB integrity (
see
Note 13
).
26. Distribute the mixture into two closeable centrifugation tubes.
27. Balance the centrifugation tubes (the difference should be
lower than 50 mg) and spin at 7,000 ×
g
for 30 min in a centri-
fuge equipped with a swinging bucket rotor. Only a centrifuge
tolerant to the presence of fl ammable solvent can be used.
28. Remove the upper organic fraction and resuspend the OBs in
the water phase.
29. Remove the rest of hexane under nitrogen fl ow for 10 min.
30. Distribute the suspension into three centrifugation tubes and
overlay with 5 mL of IB2.
μ
