Biology Reference
In-Depth Information
Microdomains
22.2.2
Method
22.2.2.1
Silica coating
The purpose of this method is to encapsulate the cells in ludox, cross-link with PAA
to form a cast around the membrane, and then lyse the cells using a Dounce
homogenizer.
10
6
cells).
Count the cells of interest (use between 5 and 50
Wash cells with PBS.
Resuspend cells in 1 mL of PMCB.
Add 5 mL of 5% ludox in a 50 mL conical tube.
Aspirate the cells into a large syringe.
Attach a 20-gauge needle and then add cells from syringe one drop at a time to the
ludox solution while manually swirling the tube.
Add PMCB to a final volume of 20 mL.
Centrifuge at 900
g
for 3 min and then aspirate the supernatant. Appearance of
a “fluffy” pellet generally means that the cells are already being lysed or
dead. This is not desired.
Wash twice with 20 mL PMCB.
Resuspend in 1 mL of PMCB.
Add the silica-coatedcells dropwise as describedpreviously to5 mLof the 1 mg/mL
PAA solution in a fresh 50 mL tube while swirling manually.
Dilute by topping up to 20 mL PMCB.
Centrifuge at 900
g
for 3 min and then discard the supernatant.
Resuspend in 2 mL of cold lysis buffer, put on ice for 30 min, and agitate
occasionally.
Lyse cells in prechilled Dounce homogenizer using the loose-fitting pestle (ten
strokes) and then the tight-fitting pestle (three strokes).
Check that cells have lysed using a light microscope.
22.2.2.2
Fractionation
Now, the plasma membrane is very dense and can be isolated from internal mem-
branes and nuclear debris.
Centrifuge silica membranes (900
g
for 3 min).
Remove fluid phase and transfer it to microfuge tubes. This contains internal
membranes and the cytosolic fraction.
Spin the fluid phase at 20,000
g
for 10 min at 4
C.
The pellet is internal membranes and the new fluid phase is cytosol.
Add a small amount of lysis buffer to the internal membranes, plus 4
sample
buffer, then boil, and use for Western blotting.
Take the cytosol phase and add 4
sample buffer, boil, and use for Western
blotting.
Resuspend the silica membranes in 11 mL of lysis buffer.
Layer this onto 2 mL of 70% Histodenz in the ultracentrifuge tube.
