title: Quantitative analysis of surface expression of membrane proteins using cold-adapted proteases.
abstract: This unit presents an improved method for quantitative analysis of surface expression of membrane proteins utilizing a cold-adapted trypsin. Preservation of the proteolytic activity of the enzyme at 0° to 4°C allows cleavage of surface-expressed membrane proteins at temperatures at which trafficking of the mammalian plasmalemmal proteins is blocked. This provides an important advantage over established trypsin-cleavage protocols since it can be applied to membrane proteins with a fast turnover rate of the membrane pool and a fast recycling rate. Compared to surface biotinylation, the method is less time consuming.
authors: Department of Biosciences and Neuroscience Center, University of Helsinki, Helsinki, Finland.
authors: Faraz
authors: F
authors: Ahmad
authors: Department of Biosciences and Neuroscience Center, University of Helsinki, Helsinki, Finland.
authors: Kai
authors: K
authors: Kaila
authors: Department of Biosciences and Neuroscience Center, University of Helsinki, Helsinki, Finland.|Institute of Physiology I, Westfälische Wilhelms-University Münster, Münster, Germany.
authors: Peter
authors: P
authors: Blaesse
mesh_terms: D000818:Animals; D019921:Biotinylation; D061505:Cell Surface Display Techniques / Q000379:methods*; D002478:Cells, Cultured; D003080:Cold Temperature; D048388:Gadiformes; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism*; D015202:Protein Engineering; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D014357:Trypsin
keywords: BS3; biotinylation; membrane protein; surface analysis; surface expression; trafficking; trypsin