Biology Reference
In-Depth Information
CHAPTER
Lipidome
2
Claudia Schmidt
1
, Birgit Ploier
1
, Barbara Koch, and G ยจ nther Daum
Institute of Biochemistry, Graz University of Technology, Graz, Austria
Analysis of Yeast Lipid
Droplet Proteome and
CHAPTER OUTLINE
Introduction .............................................................................................................. 16
2.1 Materials........................................................................................................... 18
2.1.1 Equipment and Supplies ................................................................... 18
2.1.2 Media and Reagents ......................................................................... 19
2.1.3 Databases........................................................................................ 20
2.2 Methods ............................................................................................................ 20
2.2.1 Isolation of LD From Yeast ................................................................ 20
2.2.2 Protein Analysis of LD....................................................................... 21
2.2.2.1 Protein Determination .................................................................21
2.2.2.2 Purity Control by Western Blot Analysis .......................................22
2.2.2.3 MS of Proteins............................................................................22
2.2.3 Lipid Analysis of LD.......................................................................... 23
2.2.3.1 Lipid Extraction...........................................................................23
2.2.3.2 Thin Layer Chromatographic Analysis of Nonpolar Lipids .............23
2.2.3.3 Phospholipid Analysis .................................................................24
2.2.3.4 Gas Liquid Chromatographic Analysis of FA.................................25
2.2.3.5 GLC/MS of Sterols.......................................................................25
2.2.3.6 MS of Nonpolar Lipids and Phospholipids ...................................26
2.3 Results and Discussion ...................................................................................... 26
2.3.1 SDS-PAGE and Western Blot ............................................................. 26
2.3.2 Proteome Analysis of LD ................................................................... 28
2.3.3 Lipid Analysis of LD.......................................................................... 28
2.3.4 Lipid Composition of LD From S. cerevisiae, P. pastoris, and
Y. lipolytica...................................................................................... 29
2.4 Notes ................................................................................................................ 33
Acknowledgments ..................................................................................................... 33
References ............................................................................................................... 34
1
Contributed equally to this manuscript and should be considered as co-first authors.
