The detection of phospholipase-resistant and -sensitive glycosyl-phosphatidylinositol membrane anchors by western blotting.
Glycosyl-phosphatidylinositol (GPI) membrane anchors are present on a large number of eukaryotic plasma membrane proteins. Some of these anchors can be cleaved with bacterial phosphatidylinositol-specific phospholipases C, and a glycosyl-phosphatidylinositol-specific phospholipase C from Trypanosoma brucei, to reveal an epitope called the cross-reacting determinant. Other glycosyl-phosphatidylinositol anchors are resistant to the action of these enzymes prior to treatment with mild base. A simple method is described for identifying both phospholipase-sensitive and -resistant anchors using anti-cross-reacting determinant antibodies on Western blots. This procedure represents a high-sensitivity general method for the identification of GPI-anchored proteins.
Guther
M L
ML
Department of Microbiology, Immunology, and Parasitology, Escola Paulista de Medicina, Sao Paulo, Brazil.
de Almeida
M L
ML
Rosenberry
T L
TL
Ferguson
M A
MA
D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D000939:Epitopes / Q000032:analysis; D017261:Glycosylphosphatidylinositols / Q000032:analysis* / Q000378:metabolism; D015394:Molecular Structure; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology; D010738:Type C Phospholipases / Q000378:metabolism*
D000939:Epitopes; D017261:Glycosylphosphatidylinositols; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D043265:Phosphatidylinositol Diacylglycerol-Lyase