Protocol to enrich and analyze plasma membrane proteins from frozen tissues
Methods Mol Biol. 2008;432:175-83
Jacek R. Wiśniewski
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.
http://www.ncbi.nlm.nih.gov/pubmed/18370018
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