Biology Reference
In-Depth Information
polyene-fatty acids (
Kuerschner et al., 2005
), etc. With these tools in hands, we are
able to start answering important questions regarding the mechanism of CLDs bio-
genesis, such as the formation of nascent CLDs and addition of lipids to preformed
CLDs.
This protocol covers the isolation of CLDs and LLDs from the liver of fasted
C57BL/6J mouse maintained on chow diet. Analysis protocols related to the char-
acterization of CLDs and LLDs and visualization of CLD dynamics will also be
briefly discussed.
7.1
MATERIALS
7.1.1
Isolation of CLDs
Reagents
:
1.
Phosphate-buffered saline (PBS): NaCl 137 mM, KCl 2.7 mM,
Na
2
HPO
4
2H
2
O10mM,KH
2
PO
4
2.0 mM, pH 7.4
2.
Hypotonic medium (HM): 20 mM Tris-HCl, pH 7.4, 1 mM EDTA. Prepare
fresh, keep refrigerated
3.
60% sucrose-HM: HM supplemented with 60 g/100 ml sucrose. Prepare fresh,
keep refrigerated for no more than 1 day
4.
5% sucrose-HM: HM supplemented with 5 g/100 ml sucrose. Prepare fresh,
keep refrigerated for no more than 1 day
All hypotonic media contain protease and phosphatase inhibitors at their optimal
concentrations
.
5.
Sodium dodecyl sulfate (SDS) 10% (w/v)
Other materials and equipment
:
6.
Plastic tubes
7.
Eppendorf tubes (1.5 ml)
8.
Low-speed refrigerated centrifuge with appropriate centrifuge tubes
9.
Ultracentrifuge with swinging-bucket rotor
10.
Thin-walled polycarbonate ultracentrifuge tubes
11.
Reagents and equipment for SDS-PAGE and immunoblotting
7.1.2
Isolation of LLDs
Reagents
:
1.
Tris-buffered saline (TBS): 20 mM Tris-HCl, 150 mM NaCl
2.
Homogenizationbuffer: 250 mMsucrose, 20 mMTris-HCl, pH 7.4, 1 mMEDTA
3.
Washing buffer: 20 mM Tris-HCl, 0.5 M NaCl (pH 7.4)
4.
1 mM Tris-HCl, pH 8.8
5.
Glycerol
6.
Protease inhibitor cocktail (Roche)
