Comparison of five commercial extraction kits for subsequent membrane protein profiling |
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Authors: | Stefanie Bünger Uwe J Roblick Jens K Habermann |
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Institution: | 1.Laboratory for Surgical Research, Department of Surgery,University of Lübeck, Lübeck,Lübeck,Germany;2.Department of Medical Biochemistry and Biophysics,Karolinska Institute,Stockholm,Sweden |
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Abstract: | 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. |
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Keywords: | Extraction techniques Membrane proteins Commercial kits Sub-cellular fractions Contamination Protein yield |
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