Biology Reference
In-Depth Information
12. Discard the supernatant with an aspirator. Add an appropriate
quantity of PM-suspension medium to each tube and homog-
enize the pellets with a Tefl on-glass homogenizer. Collect
the plasma membrane suspensions with a large Pasteur
pipette into ultracentrifuge tubes. Balance ultracentrifuge
tubes in pairs with PM-suspension medium. Ultracentrifuge
again at 231,000 ×
g
for 35 min.
13. Discard the supernatant with an aspirator. Add a minimal
quantity of PM-suspension medium to the plasma membrane
pellets. Homogenize the pellets with a glass rod. Transfer into
a Tefl on-glass homogenizer and homogenize well using an
electric Tefl on-glass homogenizer (moving up and down
fi ve times) with cooling on ice. Transfer into a 1.5 mL
microtube.
14. Measure the protein content using the Bradford assay (BioRad
Protein Assay Kit). Use 10
g of protein for tryptic digestion
and LC-MS/MS analysis. The remaining PM fractions should
be frozen in liquid nitrogen immediately and stored at −80 °C.
μ
Perform all steps on crushed ice (unless indicated otherwise).
3.2 Detergent-
Resistant Membrane
Extraction
1. Prepare PM with approximately 2.5 mg protein and dilute
with PM-suspension medium in an ultracentrifuge tube. After
balancing the tubes in pairs, ultracentrifuge at 231,000 ×
g
for
35 min (
see
Note 16
).
2. Add 2,000
L of PM-suspension medium in an ultracentri-
fuge tube and grind pellets with a glass rod. Transfer into a
Tefl on-glass homogenizer and homogenize well using an elec-
tric homogenizer (moving up and down fi ve times). Measure
the protein content by Bradford assay and place PM samples
with 2 mg of protein into a 35 mL swing rotor tube. Adjust
the volume to 2.7 mL by adding PM-suspension medium.
3. Add 300
μ
L of 10 % (w/v) Triton X-100 buffer and mix well
(at this point, protein:detergent ratio is 1:15). Incubate for
30 min.
4. Add 12 mL of 65 % (w/w) sucrose solution and mix well (at
this point, the fi nal concentration of sucrose is 52 %). Overlay
5 mL of 48, 35, 30, and 5 % (w/w) sucrose solution slowly in
sequence (
see
Note 17
).
5. Balance the swing rotor tubes in pairs by adding 5 % (w/w)
sucrose solution and ultracentrifuge in a swing rotor at
141,000 ×
g
for 20 h.
6. DRMs will be visible as a white layer at the interface of the
35 %/48 % (w/w) sucrose solution. Recover the white layer
and place it in an ultracentrifuge tube. Dilute with TED buffer
μ
