Biology Reference
In-Depth Information
2.
Individual mouse heart is manually homogenized using the fitted glass pestle,
and keeping the homogenizer and tissue on ice until the tissue is completely
homogenized.
3.
Homogenates are obtained and pooled from five mouse hearts together into a
50 ml conical centrifuge tube.
4.
Homogenates are centrifuged at 100 gavg. at 4
C for 10 min (Refrigerated
Centrifuge, Allegra 22 X-R), collect 10
l twice for later protein determination,
and then transfer the supernatant to a 5 ml ultracentrifuge tube. Pellets are kept
on ice to be later processed.
5.
Supernatant is centrifuged at 237,020 gavg. for 2 h at 4
C (Ultracentrifuge,
Beckman Optima).
6.
The LDs will concentrate in a thin white band at the top of the tube. LDs are
collected using a 200
m
l pipette tip with large orifice and transferred to an
Eppendorf tube, trying to obtain the most LDs with the least amount of buffer.
7.
Collect remaining infranatant, add 1:1 amount of 2
m
concentrate Laemmli gel
80
C.
8.
Wash pellets collected after the low-speed centrifugation and high-speed
centrifugation steps by resuspending pellets with 200
sample buffer solution, and freeze at
l of Tris-EDTA buffer
pH 7.4 and centrifugation at 18,000 gavg. for 30 min at 4
C (Microfuge,
Beckman 22R). Finally, pellets are solubilized in 200
m
l of Tris-EDTA buffer
pH 7.4 containing 10% SDS and 200
m
l2
sample buffer solution.
9.
LDs are separated from the remaining buffer by centrifuging at 18,000 gavg. for
30 min at 4
C (Microfuge, Beckman 22R).
10.
Working rapidly to avoid warming the layer of LDs, the solution underlying the
LDs is removed using a 1 ml syringe fitted with a 27G1/2 needle cut with a blunt
end.
11.
200
m
l of Tris-EDTA buffer pH 7.4 is added to the LDs by releasing the buffer
close to where the lipid layer is sticking to the plastic centrifuge tube and LDs
are resuspended by gentle tapping on the tube. Two 10
m
m
l aliquots of LDs
20
C to be used later for LDs
containing solution are taken and frozen at
protein determination.
12.
Proteins are then acetone-precipitated from the remaining LD containing
solution by adding 1:3 (v/v) of acetone, vortex and freeze at
20
C overnight.
13.
The following day, collect the LD acetone-precipitated proteins by centrifuging
the Eppendorf tubes for 1 h at 18,000 gavg. at 4
C and decant the tube. Let the
tube dry briefly on ice in the hood.
14.
Add 80
m
l of 10% SDS, 20
m
l of 10 mM DTT, and 100
m
lof2
concentrate
Laemmli sample buffer to solubilize the precipitated proteins.
8.1.4
Analysis of LDs by western blot
1.
Separate the LD proteins using 4-12% Bis-Tris gels according to the standard
methods.
2.
Transfer the proteins to a nitrocellulose membrane (Amersham Biosciences, NJ)
by following the standard procedures.
