SpringerProtocols: Abstract: Protocol to Enrich and Analyze Plasma Membrane Proteins from Frozen Tissues

Protocol to Enrich and Analyze Plasma Membrane Proteins from Frozen Tissues

By: Jacek R Wiśniewski⁴

Abstract

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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.

Affiliation(s): (4) Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Martinsried, Germany

Book Title: Organelle Proteomics

Series: Methods in Molecular Biology | Volume: 432 | Pub. Date: Feb-01-2008 | Page Range: 175-183 | DOI: 10.1007/978-1-59745-028-7_12

Subject: Biochemistry

Key Words: Frozen tissue - membrane proteins - plasma membrane - mouse brain

References

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