Detection of glycophospholipid anchors on proteins.
Many eukaryotic proteins are tethered to the plasma membrane by glycosyl phosphatidylinositol (GPI) membrane anchors. This unit provides a general approach for detecting GPI-anchored proteins. First, the detergent-partitioning behavior of a protein of interest is examined for characteristics of GPI-linked species. The partitioning of total cellular and isolated proteins with Triton X-114 is described in this unit, and precondensation of Triton X-114, which is necessary to remove hydrophilic contaminants before partitioning, is outlined in a Support Protocol 1. The protein may also be subjected to specific enzymatic or chemical cleavages to release it from its GPI anchor. Phospholipase cleavage (starting with intact cells or membranes, or with isolated protein) is detailed, and chemical cleavage with nitrous acid is also described. If GPI-anchored proteins are radiolabeled with fatty acids, it facilitates the detection of the GPI protein products following the cleavage reactions. A protocol for separation of lipid moieties released from proteins is provided and base hydrolysis of proteins is also presented.
Doering
T L
TL
University of California, Berkeley, California, USA.
Englund
P T
PT
Hart
G W
GW
D000818:Animals; D000906:Antibodies; D001671:Biochemistry / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D017261:Glycosylphosphatidylinositols / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D006868:Hydrolysis / Q000187:drug effects; D009608:Nitrous Acid; D017830:Octoxynol; D043265:Phosphatidylinositol Diacylglycerol-Lyase / Q000378:metabolism; D010740:Phospholipases / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D011868:Radioisotopes
D000906:Antibodies; D003902:Detergents; D017261:Glycosylphosphatidylinositols; D011506:Proteins; D011868:Radioisotopes; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D010740:Phospholipases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D009608:Nitrous Acid