Biology Reference
In-Depth Information
14. ZipTip equilibration solution: 1 % FA in water. Mix 40
μ
L of
L of water in a glass test tube. Prepare immedi-
ately before use and sonicate in a water bath for 5 min to
remove air bubbles.
15. ZipTip elute solution: 50 % ACN, 1 % FA. Mix 198
FA and 3,960
μ
μ
L of
ZipTip wash solution with 2
L of FA. Store in a glass vial (
see
Note 8
). Prepare immediately before use and sonicate in a
water bath for 5 min to remove air bubbles.
μ
2.4 Solutions
for LC-MS/MS
1. Dissolving solution: 5 % ACN, 0.1 % FA. Mix 5 mL of ACN
and 0.1 mL of FA and make up to 100 mL with water.
2. Mobile phase A: 0.1 % (v/v) FA in water. Mix 1 mL of FA and
999 mL of water.
3. Mobile phase B: 0.1 % (v/v) FA acid in ACN. Mix 1 mL of FA
and 999 mL of ACN (HPLC grade).
3
Methods
3.1 OB Isolation
1. Weigh 1 g of
Arabidopsis thaliana
grains and soak them for 1 h
in IB1 (5 mL) at 4 °C.
2. Grind the grains slowly in a mortar with a pestle for 20 min on
ice. Successively, during the grinding, add 15 mL of IB1.
3. Rinse the mortar with 5 mL of IB1 and grind the grains 15
times for 30 s with a Potter grinder driven by a Heidolph
motor (rate 7). Between each grinding cycle, the suspension
should be cooled on ice for 30 s. Rinse the grinder with 5 mL
of IB1.
4. Take away 30
L of the rude extract into an Eppendorf tube
and store at 4 °C for the control of OB isolation.
5. Distribute the suspension into six centrifugation tubes and
overlay with 5 mL of IB2.
6. Balance the centrifugation tubes (the difference should be
lower than 1 mg) and spin at 100,000 ×
g
for 1 h in an ultra-
centrifuge equipped with a swinging bucket rotor.
7. After centrifugation, fl oating OB fraction should be found on
the surface. Collect the OB fraction with a spatula (
see
Note 10
).
8. Resuspend thoroughly the collected OB fraction in 2 mL of
IB3 (
see
Note 11
).
9. Take away 30
μ
L of resuspended OBs to control the OB
isolation. Make the OB suspension up to 30 mL with IB3.
10. Distribute the suspension into six centrifugation tubes and
overlay with 5 mL of IB4.
μ
