Sample preparation project for the subcellular proteome of mouse liver.
Organelle proteome has become one of the most important fields of proteomics, and the subcellular fractionation with high purity and yield has always been a challenge for cell biologists and also for the Human Liver Proteome Project (HLPP). The liver of a C57BL/6J mouse was chosen as the model to find the optimum method for subcellular preparation. The method we selected could obtain the multiple fractions including plasma membrane, mitochondria, nucleus, ER, and cytosol from a single homogenate. With the same procedure, it is for the first time that the preparation method of frozen homogenized livers was compared with that of the fresh livers and frozen livers. We systematically evaluated the purity, efficiency, and integrity by protein yield, immunoblotting, and transmission electron microscopy. Taken together, the method of multiple fractions from a single tissue is effective enough for subcellular fractionation of mouse liver. We give a selective sample preparation method for frozen homogenized livers, for rare clinical samples, which cannot easily be used for subcellular separation immediately. But the frozen livers are not recommended for organelles isolation. This result is especially useful for sample preparation of human liver for subcellular fractionation of HLPP.
Song
Yanping
Y
Department of Genomics and Proteomics, Beijing Institute of Radiation Medicine, Beijing, P R China.
Hao
Yunwei
Y
Sun
Aihua
A
Li
Tao
T
Li
Wenrui
W
Guo
Lihai
L
Yan
Yujuan
Y
Geng
Chao
C
Chen
Ning
N
Zhong
Fan
F
Wei
Handong
H
Jiang
Ying
Y
He
Fuchu
F
D000818:Animals; D006801:Humans; D008099:Liver / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D015388:Organelles / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000648:ultrastructure; D040901:Proteomics / Q000379:methods*; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure
D020543:Proteome