Protocol to enrich and analyze plasma membrane proteins.
This chapter describes a procedure for isolation and analysis of fractions enriched in plasma membranes from minute amounts of 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 of a stepwise depletion of nonintegral membrane molecules from entire tissue homogenate by high-salt, carbonate, and urea washes followed by a treatment of the membranes with sublytic concentrations of a detergent and enrichment of the plasma membranes by a density gradient fractionation. Fluorometric assays for protein content and plasma membrane marker activity allow calculation of the yield and extent of plasma membrane enrichment. Reduction, carboxymethylation, and digestion with endoproteinase Lys-C are carried out on nonsolubilized membranes. The entire procedure allows processing and preparation of samples from 10-20 mg tissue, and therefore, can be extremely helpful for proteomic profiling of biopsy-size clinical samples.
Wiśniewski
R Jacek
RJ
Department of Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Martinsried, Germany.
D002254:Carbonates / Q000737:chemistry; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D005591:Chemical Fractionation / Q000379:methods; D003902:Detergents / Q000737:chemistry; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism; D012492:Salts / Q000737:chemistry; D018401:Sample Size; D014508:Urea / Q000737:chemistry
D002254:Carbonates; D003902:Detergents; D008565:Membrane Proteins; D012492:Salts; D014508:Urea; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase