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 protein may also be subjected to specific enzymatic or chemical cleavages to release the protein from its GPI anchor. Protocols for phospholipase cleavage and chemical cleavage with nitrous acid are provided for this purpose. If GPI-anchored proteins are radiolabeled with fatty acids, it facilitates the detection of the GPI protein products following the cleavage reactions. Separation of lipid moieties and base hydrolysis of proteins are detailed herein.
Doering
T L
TL
University of California, Berkeley, California, USA.
Englund
P T
PT
Hart
G W
GW
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006017:Glycolipids / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D017942:Nitric Acid / Q000378:metabolism; D017830:Octoxynol; D010743:Phospholipids / Q000032:analysis* / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*
D006017:Glycolipids; D010743:Phospholipids; D011506:Proteins; D011092:Polyethylene Glycols; D017942:Nitric Acid; D017830:Octoxynol; C010615:Nonidet P-40