Biomedical Engineering Reference
In-Depth Information
4 Prepare one 50ml ultracentrifuge tube per 15ml lysates for iodixanol gradients through
a12cm
3
syringe connected to a 19-GA needle and 100
μ
l micro-capillary pipettes sealed
by parafilm as follows:
(a) Pour the supernatant from step 3 into the 50ml ultracentrifuge tube.
(b) Begin building the iodixanol gradient in the following order:
g
∼
15ml cell lysate from step (a)
7.5ml of of 15% iodoxanol
7.5ml of 40% iodixanol
5.0ml of 60% iodixanol
15%
h
25%
40%
60%
Iodixanol 60% (ml)
12.50
20.84
33.25
50
1
×
PBS-MK (ml)
27.50
29.16
16.75
0
5
M
NaCl (ml)
10.0
0
0
Phenol red (
μ
l)
0
100
0
125
5 Clean top and remove liquid in the neck of the ultracentrifuge tube by
Kimwipes.
6 Add lysis buffer into the top of the ultracentrifuge tube to balance the tubes.
i
7 Seal the tubes with heat sealer.
j
8 Centrifuge at 18
◦
C, 64 000
g
(69 000 rpm)in Optima L-90K ultracentrifuge with 70Ti
rotor.
9 Using a 16-GA needle connected to a 12 cm
3
syringe, carefully remove the
virus-containing fractions (about 4 ml of 40% iodixanol and 3 ml of 60% iodixanol)
k
into
a 15 ml tube and store at
−
20
◦
C for further purification by column
chromatography.
Notes
f
Each time: freezing for 10min and thawing for 15min, shake vigorously.
g
Mix the iodixanol fractions thoroughly before adding.
h
The 15% iodixanol fraction contains 1
M
NaCl to destabilize ionic interaction between
macromolecules.
i
Add lysis buffer slowly and carefully; do not interfere with the gradients.
j
To make sure the tubes are sealed completely without leaking.
k
Do not take any of inter-phase between 40% iodixanol and 25% iodixanol.
