Biology Reference
In-Depth Information
31. 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.
32. Collect the OB fl oating fraction with a spatula.
33. Resuspend thoroughly the collected OB fraction in 2 mL of IB1.
34. Take away 30
L of resuspended OBs to control the OB isola-
tion and make the OB suspension up to 10 mL with IB1.
35. Remove the rest of hexane under nitrogen fl ow for 10 min.
36. Distribute the suspension into three centrifugation tubes and
overlay with 5 mL of IB2.
37. 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.
38. Collect the OB fl oating fraction with a spatula.
39. Resuspend the collected purifi ed OBs in 500
μ
μ
L of storage
buffer.
40. To quantify the isolated OB, take away 1
L of OB suspension,
dilute it 1,000 times with the storage buffer, and measure the
OD
600
. The fi nal OD
600
of 1,000 times diluted sample should
be 0.2-0.3. Usually, the yield of the OB isolation is between
700 and 1,200
μ
L of OB suspension. Store isolated OBs at
4 °C, but no longer than 3 days.
μ
3.2
SDS-PAGE of OBs
1. Prepare the solution for 15 % SDS-PAGE gel mixing 2.3 mL
of water, 5.0 mL of 30 % AA/BIS, 2.5 mL of resolving gel buf-
fer, and 100
μ
L of 10 % SDS in a 50 mL glass beaker. Finally,
L of TEMED and slightly mix.
2. Cast quickly two gels within a 7.25 cm × 10 cm × 1.0 mm
cassette. Leave about 1.5 cm wide spaces for stacking gel and
overlay with water.
3. When the gels solidify, remove covering water.
4. Prepare stacking gel mixing 2.7 mL of water, 0.67 mL of 30 %
AA/BIS, and 0.5 mL of water in a 25 mL glass beaker. Add
40
add 100
μ
L of 10 % APS and 4
μ
μ
L of 10 % SDS. Finally, add 40
μ
L of 10 % APS and 4
μ
L
of TEMED and slightly mix.
5. Cast the stacking gels and immediately insert 10-well gel
combs without introducing air bubbles.
6. SDS-PAGE can be used either to evaluate the quality of OB
isolation, or, mainly, for further LC-MS/MS analysis.
7. To evaluate the quality of OB isolation, mix 15
L of control
samples (one rude extract, fi ve samples from purifi ed fractions,
and fi nal OB suspension) with 15
μ
L of PLB and proceed as
follows from
step 10
. The fi nal OB protein profi le is in Fig.
1
.
μ
