Biology Reference
In-Depth Information
CHAPTER
7
Huajin Wang
*
, Ariel D. Quiroga
{
, and Richard Lehner
{
Analysis of Lipid Droplets
in Hepatocytes
*
Department of Cell Biology, Yale University School of Medicine, New Haven,
Connecticut, USA
{
Instituto de Fisiologı´a Experimental, Consejo Nacional de Investigaciones Cientı´ficas
yT ´cnicas (CONICET), Facultad de Ciencias Bioquı´micas y Farmace´uticas, Universidad Nacional
de Rosario, Rosario, Argentina
{
Group on Molecular and Cell Biology of Lipids, Departments of Pediatrics and Cell Biology,
University of Alberta, Edmonton, Alberta, Canada
CHAPTER OUTLINE
Introduction and Rationale....................................................................................... 108
7.1 Materials......................................................................................................... 111
7.1.1 Isolation of CLDs ............................................................................ 111
7.1.2 Isolation of LLDs ............................................................................ 111
7.1.3 Use of BODIPY Fatty Acids to Visualize CLD Dynamics ...................... 112
7.2 Methods .......................................................................................................... 113
7.2.1 Isolation of CLDs from Mouse Liver .................................................. 113
7.2.1.1 Mice, Diets, and Feeding States ............................................... 113
7.2.1.2 CLD Fractionation .................................................................... 113
7.2.1.3 Solubilization of CLD-Associated Proteins for Immunoblotting ... 115
7.2.2 Isolation of LLDs from Mouse Liver .................................................. 115
7.2.2.1 Preparation of the Mouse......................................................... 115
7.2.2.2 Isolation of the Liver and Tissue Homogenization...................... 115
7.2.2.3 Isolation of Microsomes ........................................................... 116
7.2.2.4 Release of Microsomal Lumenal Content .................................. 117
7.2.2.5 Separation of LLDs from VLDL Precursors ................................ 117
7.2.2.6 Isolation of Microsomal LLDs by Density Gradient
Ultracentrifugation................................................................................ 117
7.2.2.7 Sample Analysis ...................................................................... 118
