Comparison of five commercial extraction kits for subsequent membrane protein profiling.
Membrane proteins account for 70-80% of all pharmaceutical targets emphasizing their clinical relevance. Identification of new, differentially expressed membrane proteins reflecting distinct disease properties is thus of high importance. Unfortunately, isolation and analysis of membrane-bound proteins is hampered by their relative low abundance in total cell lysates, their frequently large size and their hydrophobic properties. We thus aimed to identify protocols that allow for highly efficient isolation and purification of membrane-bound proteins for subsequent protein profiling. We present a comparative study of different membrane protein extraction methods that vary in total protein yield between 0.02 and 4.8 mg using constant cell pellets of the colorectal carcinoma cell line SW620. We also demonstrate by means of polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis that the majority of commercial membrane extraction kits harbor a substantial cytosolic contamination of their membranous fraction. Based on purity of membranous fraction, protein yield, time and costs, we show superiority of two commercial extraction kits for downstream proteome analyses of membrane proteins.
Bünger
Stefanie
S
Laboratory for Surgical Research, Department of Surgery, University of Lübeck, Lübeck, Ratzeburger Allee 160, 23538, Lübeck, Germany.
Roblick
Uwe J
UJ
Habermann
Jens K
JK