Protocol to enrich and analyze plasma membrane proteins from frozen tissues.
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.
Wiśniewski
Jacek R
JR
Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Martinsried, Germany.
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
D007202:Indicators and Reagents; D008565:Membrane Proteins