title: Protocol to enrich and analyze plasma membrane proteins from frozen tissues.
abstract: This chapter presents procedures for preparation and analysis of fractions enriched in plasma membranes from frozen tissue. It consists of a method for extraction and fractionation of membranes and a method for enzymatic digestion of membrane proteins without use of detergents. The method for isolation of membranes comprises a stepwise depletion of non-integral membrane proteins from entire tissue homogenate by high-salt, carbonate, and urea washes followed by treatment of the membranes with sublytic concentrations of digitonin and enrichment of the plasma membranes by a density gradient fractionation. Reduction, carboxymethylation, and digestion with endoproteinase Lys-C are carried out on non-solubilized membranes. The entire procedure allows processing and preparation of samples of minute amounts as 10-20 mg tissue and therefore can be extremely helpful for proteomic profiling of small pieces of tissue and clinical material.
authors: Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Martinsried, Germany.
authors: Jacek R
authors: JR
authors: Wiśniewski
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002462:Cell Membrane / Q000648:ultrastructure*; D002499:Centrifugation, Density Gradient / Q000379:methods; D005612:Freeze Drying; D007202:Indicators and Reagents; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification; D051379:Mice
chemical_list: D007202:Indicators and Reagents; D008565:Membrane Proteins