Purification and proteomic analysis of liver membrane skeletons.
The detergent-resistant membrane skeletons play a critical role in cell shaping and signaling. The focus of the methods described in this chapter is first on the preparation of membrane skeletons from liver by multistep sucrose density gradient centrifugation, and then on the analysis of the protein components of membrane skeletons using proteomics techniques. Two proteomic analysis strategies are described. In the first strategy, membrane skeleton proteins are separated by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. In the other strategy, proteins are separated by SDS-PAGE and identified by liquid chromatography-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. The methods facilitate the understanding of the structure of membrane skeletons.
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, People's Republic of China.
Jintang
J
He
Yashu
Y
Liu
Qingsong
Q
Wang
Jianguo
J
Ji
D000818:Animals; D015153:Blotting, Western; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D003902:Detergents / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008099:Liver / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D017830:Octoxynol / Q000737:chemistry; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D014357:Trypsin / Q000737:chemistry
D003598:Cytoskeletal Proteins; D003902:Detergents; D020543:Proteome; D017830:Octoxynol; D014357:Trypsin